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Sample records for 10-formyl tetrahydrofolate deformylases

  1. Single-stranded DNA binding activity of C1-tetrahydrofolate synthase enzymes.

    PubMed

    Wahls, W P; Song, J M; Smith, G R

    1993-11-15

    In eukaryotes C1-5,6,7,8-tetrahydrofolate (THF) synthase is a trifunctional enzyme that catalyzes the interconversion of reduced forms of folate to supply activated one-carbon units required for a variety of metabolic pathways. The enzymatic activities include 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). In bacteria separate, monofunctional or bifunctional polypeptides catalyze the same reactions. We have purified C1-THF synthase from the fission yeast Schizosaccharomyces pombe and found its physical and enzymatic properties similar to those of other eukaryotic C1-THF synthase enzymes. Unexpectedly, the S. pombe enzyme bound strongly (Keq = 100 pM) to single-stranded DNA, but not to double-stranded DNA or to RNA. The binding was sequence-independent, apparently not cooperative, and not detectably inhibited by C1-THF synthase substrates or cofactors. Trifunctional cytoplasmic enzyme from Saccharomyces cerevisiae and monofunctional (synthetase) enzyme from Clostridium acidiurici also bound tightly to single-stranded DNA, while bifunctional (dehydrogenase and cyclohydrolase) enzyme from Escherichia coli did not, suggesting that single-stranded DNA binding is a conserved function of the synthetase domain of C1-THF synthase enzymes. PMID:8226914

  2. 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.

  3. Therapeutic potential of peptide deformylase inhibitors.

    PubMed

    Chen, D; Yuan, Z

    2005-09-01

    Peptide deformylase (PDF) is an attractive target for antibacterial drug discovery. Progress in the biological characterisation of the enzyme, coupled with newly obtained mechanistic and structural insight, enabled the pharmaceutical industry to discover potent PDF inhibitors that can be considered as clinical development candidates for this new class of antibacterial agents. The in vitro and in vivo data for several lead PDF inhibitors suggest that the current PDF inhibitors are most suitable for the treatment of respiratory tract infections and they are not cross-resistant to the current clinically used antibiotics. Two PDF inhibitors, BB-83698 and VIC-104959, have progressed to Phase I clinical trials by intravenous and oral administration, respectively. Both of these compounds show promising in vitro and in vivo efficacy and an excellent safety profile. The pharmacokinetics in humans for both of the compounds suggest the possibility of a twice-daily dosing regimen for clinical use. Thus far, all of the data suggest a promising future for this new class of antibacterial agents.

  4. Comparative QSAR studies on peptide deformylase inhibitors.

    PubMed

    Lee, Ji Young; Doddareddy, Munikumar Reddy; Cho, Yong Seo; Choo, Hyunah; Koh, Hun Yeong; Kang, Jae-Hoon; No, Kyoung Tai; Pae, Ae Nim

    2007-05-01

    Comparative quantitative structure-activity relationship (QSAR) analyses of peptide deformylase (PDF) inhibitors were performed with a series of previously published (British Biotech Pharmaceuticals, Oxford, UK) reverse hydroxamate derivatives having antibacterial activity against Escherichia coli PDF, using 2D and 3D QSAR methods, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and hologram QSAR (HQSAR). Statistically reliable models with good predictive power were generated from all three methods (CoMFA r (2) = 0.957, q (2) = 0.569; CoMSIA r (2) = 0.924, q (2) = 0.520; HQSAR r (2) = 0.860, q (2) = 0.578). The predictive capability of these models was validated by a set of compounds that were not included in the training set. The models based on CoMFA and CoMSIA gave satisfactory predictive r (2) values of 0.687 and 0.505, respectively. The model derived from the HQSAR method showed a low predictability of 0.178 for the test set. In this study, 3D prediction models showed better predictive power than 2D models for the test set. This might be because 3D information is more important in the case of datasets containing compounds with similar skeletons. Superimposition of CoMFA contour maps in the active site of the PDF crystal structure showed a meaningful correlation between receptor-ligand binding and biological activity. The final QSAR models, along with information gathered from 3D contour and 2D contribution maps, could be useful for the design of novel active inhibitors of PDF. PMID:17333308

  5. 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

  6. Peptide deformylase of eukaryotic protists: a target for new antiparasitic agents?

    PubMed

    Meinnel, T

    2000-04-01

    Peptide deformylase is found only in Eubacteria, making it a logical target for discovering new antibacterial agents. Although this protein is absent from animal or fungal cells, evidence supports its existence in eukaryotic protists, including the causative agents of malaria, sleeping sickness, Chagas disease and leishmaniosis. Here, Thierry Meinnel discusses the idea that deformylase inhibitors could be used as very broad-spectrum antibiotics against bacterial infections, as well as parasitic diseases.

  7. 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

  8. Investigating Deformylase and Deacylase Activity of Mammalian and Bacterial Sirtuins.

    PubMed

    Seidel, Julian; Klockenbusch, Cordula; Schwarzer, Dirk

    2016-03-01

    Lysine acylation constitutes a major group of post-translational modifications of proteins, and is found in the proteomes of organisms from all kingdoms of life. Sirtuins are considered the main erasers of these modification marks, and thus contribute to acylation-dependent regulation of enzyme activity, and potentially of protein quality control. We have established a substrate scaffold to enable the analysis of sirtuin activity with a broad range of acyl-lysine modifications, including hydrophobic fatty acids. Characterization of the deacylase activity of the bacterial SrtN, which is encoded by the yhdZ gene of Bacillus subtilis, showed that this enzyme is capable of removing a broad range of acyl groups. These investigations further showed that SrtN and human SIRT1 are efficient lysine-deformylases, thereby providing a first clue as to how this nonenzymatic modification might be removed from affected proteins.

  9. A eubacterial riboswitch class that senses the coenzyme tetrahydrofolate.

    PubMed

    Ames, Tyler D; Rodionov, Dmitry A; Weinberg, Zasha; Breaker, Ronald R

    2010-07-30

    Comparative sequence analyses of bacterial genomes are revealing many structured RNA motifs that function as metabolite-binding riboswitches. We have identified an RNA motif frequently positioned in the 5' UTRs of folate transport and biosynthesis genes in Firmicute genomes. Biochemical experiments confirm that representatives of this new-found RNA class selectively bind derivatives of the vitamin folate, including di- and tetrahydrofolate coenzymes. In addition, representatives of this aptamer class occasionally reside upstream of RNA structures that are predicted to control translation initiation in response to ligand binding. These findings expand the number of coenzymes that are directly sensed by RNA and reveal possible riboswitch-controlled regulons that respond to changes in single-carbon metabolism.

  10. Compartmentation of cellular tetrahydrofolate pools in rat liver

    SciTech Connect

    Barlowe, C.K.; Appling, D.R.

    1987-05-01

    Folate coenzymes are essential in shuttling one carbon units between a number of cellular processes. Serine, glycine, histidine and formate may serve as one-carbon sources by donation of their one-carbon unit to tetrahydrofolate (THF) which may then be utilized in the biosynthesis of nucleic acids and amino acids or oxidized to carbon dioxide (CO/sub 2/) via formyl-THF dehydrogenase. Nitrous Oxide (N/sub 2/O) has been observed to inactivate methionine synthase causing an increase in the cellular concentration of methyl-THF at the expense of other folate forms, specifically THF. They are interested in determining if THF dependent pathways that utilize serine, formate and histidine are equally effected by N/sub 2/O. Radiometric methods indicate that oxidation of the ..beta..-carbon of serine to CO/sub 2/ is uneffected by N/sub 2/O exposure, but oxidation of formate and the 2-ring carbon of histidine to CO/sub 2/ are reduced by about 50% and 95% of control levels, respectively. In contrast, the use of these one carbon sources for purine biosynthesis are all reduced roughly 50% upon exposure to N/sub 2/O. These data suggest an intracellular compartmentation of THF pools available to different sources of one-carbon units.

  11. 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

  12. 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

  13. The role of peptide deformylase in protein biosynthesis: a proteomic study.

    PubMed

    Bandow, Julia Elisabeth; Becher, Dörte; Büttner, Knut; Hochgräfe, Falko; Freiberg, Christoph; Brötz, Heike; Hecker, Michael

    2003-03-01

    Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase. The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible. Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point. Two strategies were used to investigate the nature of the charge shift. In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared. Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation. Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type. The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type. Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift. Eight of these protein pairs were also present on 2-D gels of exponentially growing B. subtilis, where the more acidic, still formylated protein species represented the smaller parts.

  14. A unique peptide deformylase platform to rationally design and challenge novel active compounds

    PubMed Central

    Fieulaine, Sonia; Alves de Sousa, Rodolphe; Maigre, Laure; Hamiche, Karim; Alimi, Mickael; Bolla, Jean-Michel; Taleb, Abbass; Denis, Alexis; Pagès, Jean-Marie; Artaud, Isabelle; Meinnel, Thierry; Giglione, Carmela

    2016-01-01

    Peptide deformylase (PDF) is considered an excellent target to develop antibiotics. We have performed an extensive characterization of a new PDF from the pathogen Streptococcus agalactiae, showing properties similar to other known PDFs. S. agalactiae PDF could be used as PDF prototype as it allowed to get complete sets of 3-dimensional, biophysical and kinetic data with virtually any inhibitor compound. Structure-activity relationship analysis with this single reference system allowed us to reveal distinct binding modes for different PDF inhibitors and the key role of a hydrogen bond in potentiating the interaction between ligand and target. We propose this protein as an irreplaceable tool, allowing easy and relevant fine comparisons between series, to design, challenge and validate novel series of inhibitors. As proof-of-concept, we report here the design and synthesis of effective specific bacterial PDF inhibitors of an oxadiazole series with potent antimicrobial activity against a multidrug resistant clinical isolate. PMID:27762275

  15. Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Thermus thermophilus HB8.

    PubMed

    Kamo, Masayuki; Kudo, Norio; Lee, Woo Cheol; Motoshima, Hiroyuki; Tanokura, Masaru

    2004-07-01

    Peptide deformylase (PDF) is responsible for cleaving the formyl group at the N-terminus of nascent polypeptide chains in eubacteria and is essential to bacterial cell viability. A recombinant PDF of the thermophilic bacterium Thermus thermophilus HB8 has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals belonged to the tetragonal space group P4(1) or P4(3), with unit-cell parameters a = b = 62.58, c = 105.27 A, and are most likely to contain two molecules in an asymmetric unit, giving a crystal volume per protein weight (V(M)) of 2.3 A(3) Da(-1) and a solvent content of 46.7%.

  16. Disk diffusion quality control guidelines for NVP-PDF 713: a novel peptide deformylase inhibitor.

    PubMed

    Anderegg, Tamara R; Jones, Ronald N

    2004-01-01

    NVP-PDF713 is a peptide deformylase inhibitor that has emerged as a candidate for treating Gram-positive infections and selected Gram-negative species that commonly cause community-acquired respiratory tract infections. This report summarizes the results of a multi-center (seven participants) disk diffusion quality control (QC) investigation for NVP PDF-713 using guidelines of the National Committee for Clinical Laboratory Standards and the standardized disk diffusion method. A total of 420 NVP-PDF 713 zone diameter values were generated for each QC organism. The proposed zone diameter ranges contained 97.6-99.8% of the reported participant results and were: Staphylococcus aureus ATCC 25923 (25-35 mm), Streptococcus pneumoniae ATCC 49619 (30-37 mm), and Haemophilus influenzae ATCC 49247 (24-32 mm). These QC criteria for the disk diffusion method should be applied during the NVP-PDF 713 clinical trials to maximize test accuracy.

  17. 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

  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. 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.

  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. 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

  2. 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

  3. 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.

  4. Pharmacokinetics and unexpected safety issues of LBM415, a novel oral peptide deformylase inhibitor.

    PubMed

    Rolan, P; Sun, H; Macleod, C; Bracken, K; Evans, T G

    2011-08-01

    Peptide deformylase (PDF) inhibitors represent a potential new class of antibiotics targeting a large number of bacterial species. We studied the pharmacokinetics and safety of LBM415, a novel PDF inhibitor, administered as a single oral dose at 100-3,000 mg in the fasted state and at 1,000 mg in the fed state in healthy volunteers. LBM415 was then administered at dosages ranging from 100 mg q.d. to 1,000 mg t.i.d. for 11 days. Dose-proportional pharmacokinetics was observed, with a peak plasma concentration (C(max)) of 17.85 ± 5.96 µg/ml at 1,000 mg b.i.d. (the projected therapeutic dose) and an area under the concentration-time curve (AUC)(0-24h) of 36.83 ± 10.36 µg/ml·h. The half-life, as determined after a 1,000-mg single dose, was 2.18 ± 0.61 h. The compound was well tolerated at low doses, but at the highest dose, 1,000 mg t.i.d., reversible cyanosis and low oxygen saturation, attributable to methemoglobinemia, were detected on day 11. Oxygen saturation was as low as 88% in one subject on day 11.

  5. In vitro and ex vivo activity of peptide deformylase inhibitors against Mycobacterium tuberculosis H37Rv.

    PubMed

    Sharma, Anshika; Sharma, Sadhna; Khuller, G K; Kanwar, A J

    2009-09-01

    Bacterial peptide deformylase (PDF) catalyses removal of the N-terminal formyl group of proteins and is essential for protein maturation, growth and survival of bacteria. Thus, PDF appears to be a good antimycobacterial drug target. In the present study, various well-known PDF inhibitors, such as BB-3497, actinonin, 1,10-phenanthroline, hydroxylamine hydrochloride and galardin, were selected to evaluate their inhibitory activity against Mycobacterium tuberculosis. All compounds were found to be active against M. tuberculosis, with MIC(90) values (lowest drug concentration at which 90% of growth was inhibited on the basis of CFU enumeration) ranging from 0.2 mg/L to 74 mg/L. BB-3497 and 1,10-phenanthroline exhibited potent in vitro antimycobacterial activity, and also showed synergism with isoniazid and rifampicin. All compounds showed a bacteriostatic mode of inhibition. Under ex vivo conditions and short-course chemotherapy, BB-3497 and actinonin were found to be significantly active, with BB-3497 exhibiting comparable efficacy to that of isoniazid. Collectively, promising activities of PDF inhibitors such as BB-3497 and actinonin suggest their potential use against M. tuberculosis.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

    PubMed Central

    ANTCZAK, CHRISTOPHE; SHUM, DAVID; ESCOBAR, SINDY; BASSIT, BHRAMDEO; KIM, EARL; SESHAN, VENKATRAMAN E.; WU, NIAN; YANG, GUANGLI; OUERFELLI, OUATHEK; LI, YUE-MING; SCHEINBERG, DAVID A.; DJABALLAH, HAKIM

    2008-01-01

    The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid–based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)–based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent. PMID:17435169

  11. Promiscuous and adaptable enzymes fill "holes" in the tetrahydrofolate pathway in Chlamydia species.

    PubMed

    Adams, Nancy E; Thiaville, Jennifer J; Proestos, James; Juárez-Vázquez, Ana L; McCoy, Andrea J; Barona-Gómez, Francisco; Iwata-Reuyl, Dirk; de Crécy-Lagard, Valérie; Maurelli, Anthony T

    2014-07-08

    Folates are tripartite molecules comprising pterin, para-aminobenzoate (PABA), and glutamate moieties, which are essential cofactors involved in DNA and amino acid synthesis. The obligately intracellular Chlamydia species have lost several biosynthetic pathways for essential nutrients which they can obtain from their host but have retained the capacity to synthesize folate. In most bacteria, synthesis of the pterin moiety of folate requires the FolEQBK enzymes, while synthesis of the PABA moiety is carried out by the PabABC enzymes. Bioinformatic analyses reveal that while members of Chlamydia are missing the genes for FolE (GTP cyclohydrolase) and FolQ, which catalyze the initial steps in de novo synthesis of the pterin moiety, they have genes for the rest of the pterin pathway. We screened a chlamydial genomic library in deletion mutants of Escherichia coli to identify the "missing genes" and identified a novel enzyme, TrpFCtL2, which has broad substrate specificity. TrpFCtL2, in combination with GTP cyclohydrolase II (RibA), the first enzyme of riboflavin synthesis, provides a bypass of the first two canonical steps in folate synthesis catalyzed by FolE and FolQ. Notably, TrpFCtL2 retains the phosphoribosyl anthranilate isomerase activity of the original annotation. Additionally, we independently confirmed the recent discovery of a novel enzyme, CT610, which uses an unknown precursor to synthesize PABA and complements E. coli mutants with deletions of pabA, pabB, or pabC. Thus, Chlamydia species have evolved a variant folate synthesis pathway that employs a patchwork of promiscuous and adaptable enzymes recruited from other biosynthetic pathways. Importance: Collectively, the involvement of TrpFCtL2 and CT610 in the tetrahydrofolate pathway completes our understanding of folate biosynthesis in Chlamydia. Moreover, the novel roles for TrpFCtL2 and CT610 in the tetrahydrofolate pathway are sophisticated examples of how enzyme evolution plays a vital role in the

  12. 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.

  13. 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

  14. 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

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

    PubMed

    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

    2008-08-01

    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 A (1 A=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(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

    2014-12-12

    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.

  2. 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.

  3. 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

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

    PubMed

    Naithani, Manisha; 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.

  5. 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

  6. Equivalent absorption and in vivo kinetics of tritiated folic acid and 5-formyl-tetrahydrofolic acid in rats

    SciTech Connect

    Bhandari, S.D.; Gregory, J.F. )

    1990-02-26

    The intestinal absorption and in vivo turnover kinetics of ({sup 3}H)folic acid (FA) and (6S)-5-formyl-({sup 3}H)tetrahydrofolate (5-CHO-THF) were examined to determine whether differences exist in the inherent bioavailability of these forms of the vitamin. Following oral administration of 2 {mu}Ci/100 g body weight (in 50 mM sodium ascorbate, pH 7), a biphasic pattern of urinary tritium excretion was observed for each labeled folate. The following kinetic results were obtained (n=9). Little tritium was found in the GI tract after 8 hours, which indicated nearly complete absorption of each folate. HPLC analysis of urine revealed similar excretory patterns over 0-8 days post-dose for each folate administered, and the patterns of hepatic ({sup 3}H)folates were equivalent when examined after 8 hours and 4 days post-dose. These findings indicate that the bioavailability FA and 5-formyl-THF is equivalent.

  7. 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.

  8. Peptide deformylase inhibitor actinonin reduces celastrol’s HSP70 induction while synergizing proliferation inhibition in tumor cells

    PubMed Central

    2014-01-01

    Background Celastrol is a promising anti-tumor agent, yet it also elevates heat shock proteins (HSPs), especially HSP70, this effect believed to reduce its anti-tumor effects. Concurrent use of siRNA to increase celastrol’s anti-tumor effects through HSP70 interference has been reported, but because siRNA technology is difficult to clinically apply, an alternative way to curb unwanted HSP70 elevation caused by celastrol treatment is worth exploring. Methods In this work, we explore three alternative strategies to control HSP70 elevation: (1) Searching for cancer cell types that show no HSP70 elevation in the presence of celastrol (thus recommending themselves as suitable targets); (2) Modifying HSP70-inducing chemical groups, i.e.: the carboxyl group in celastrol; and (3) Using signaling molecule inhibitors to specifically block HSP70 elevation while protecting and/or enhancing anti-tumor effects. Results The first strategy was unsuccessful since celastrol treatment increased HSP70 in all 7 of the cancer cell types tested, this result related to HSF1 activation. The ubiquity of HSF1 expression in different cancer cells might explain why celastrol has no cell-type limitation for HSP70 induction. The second strategy revealed that modification of celastrol’s carboxyl group abolished its ability to elevate HSP70, but also abolished celastrol’s tumor inhibition effects. In the third strategy, 11 inhibitors for 10 signaling proteins reportedly related to celastrol action were tested, and five of these could reduce celastrol-caused HSP70 elevation. Among these, the peptide deformylase (PDF) inhibitor, actinonin, could synergize celastrol’s proliferation inhibition. Conclusions Concurrent use of the chemical agent actinonin could reduce celastrol’s HSP70 elevation and also enhance proliferation inhibition by celastrol. This combination presents a novel alternative to siRNA technology and is worth further investigation for its potentially effective anti

  9. Effect of H2 blockade and food on single-dose pharmacokinetics of GSK1322322, a peptide deformylase inhibitor antibacterial.

    PubMed

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

    2013-06-01

    GSK1322322 is first in a new class of antibiotics, peptide deformylase inhibitors, and is active against multidrug-resistant respiratory and skin pathogens. Part 1 of this phase 1, randomized, single-dose (1,000 mg) study in 20 healthy volunteers compared the relative bioavailability of three different tablet formulations of GSK1322322 (fast release, intermediate release, and slow release) to that of the previously studied powder-in-bottle formulation to assess the optimal formulation for progression into clinical trials. Part 2 assessed the effect of a high-fat meal and drug interaction with an H2 blocker and an H2 blocker plus vitamin C on the pharmacokinetic profile of GSK1322322. Of the three tablet formulations, fast-release GSK1322322 provided pharmacokinetic profiles similar to those of the powder-in-bottle reference formulation (~93% relative bioavailability) and was selected for progression in part 2. When GSK1322322 was administered with a high-fat meal, the maximum observed plasma concentration (C(max)) was reduced by 20%, and the time to maximum plasma concentration (T(max)) was delayed by 1.9 h. The exposure (area under the concentration-time curve [AUC]) increased by ~20% compared to that in volunteers in the fasted state. Coadministration of GSK1322322 with an H2 blocker resulted in a slight delay in absorption (T(max) ~0.75 h later) and 58 and 38% decreases in the C(max) and AUC0-∞ values, respectively, compared to GSK1322322 alone. This effect was reversed with vitamin C intake (i.e., no delay in T(max) and the C(max) and AUC0-∞ values decreased by only 21 and 12%, respectively). GSK1322322 was generally well tolerated, and most adverse events were mild in intensity during both parts of the study. PMID:23529727

  10. 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

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

    PubMed Central

    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

    2015-01-01

    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

  12. 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).

  13. 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.

  14. 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.

  15. [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.

  16. A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate

    PubMed Central

    Masai, Eiji; Sasaki, Miyuki; Minakawa, Yasunori; Abe, Tomokuni; Sonoki, Tomonori; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2004-01-01

    Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H4folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H4folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H4folate, forming 5-methyl-H4folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H4folate. The possible role of ligH is discussed. PMID:15090517

  17. A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate.

    PubMed

    Masai, Eiji; Sasaki, Miyuki; Minakawa, Yasunori; Abe, Tomokuni; Sonoki, Tomonori; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2004-05-01

    Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H(4)folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H(4)folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H(4)folate, forming 5-methyl-H(4)folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H(4)folate. The possible role of ligH is discussed.

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

    PubMed Central

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

    2011-01-01

    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

  19. Methylation pattern of methylene tetrahydrofolate reductase and small nuclear ribonucleoprotein polypeptide N promoters in oligoasthenospermia: a case-control study.

    PubMed

    Botezatu, Anca; Socolov, Razvan; Socolov, Demetra; Iancu, Iulia Virginia; Anton, Gabriela

    2014-02-01

    Alterations in DNA methylation patterns in several genes may lead to abnormal male sexual development and infertility. This study investigated the promoter methylation status of MTHFR and SNRPN in infertile men from Romania by quantitative methylation-specific PCR in order to investigate possible correlations with sperm abnormalities. The study groups included patients (n=27) with a median age of 31 years (range 26-41 years) as well as controls (n=11) with a median age of 30 years (range 24-37 years) recruited from couples seeking advice for infertility. DNA was isolated from sperm samples and promoter methylation was assessed using direct. Significant trends were detected for both genes that indicate a tendency towards promoter hypermethylation in spermatozoa with low motility (MTHFR P=0.0032, r=0.23; SNRPN P=0.0003, r=0.32) and poor morphology (MTHFR P=0.0012, r=0.27; SNRPN P=0.0003, r=0.33) but no trend was found in cases of low sperm count (MTHFR r=0.007; SNRPN r=0.06). The data indicate that the methylation patterns of the promoters of MTHFR and SNRPN are associated with changes in sperm motility and morphology, which could lead to male infertility. A large number of studies are now focused on the causes of male infertility. Among these are epigenetic modifications, which are important contributors to reproductive pathology in the male by providing dynamic changes of the phenotype according to the environmental and metabolic factors. The most known epigenetic modification is DNA methylation and alterations in this pattern in several genes could induce male infertility. The present study aims to investigate the promoter methylation status of the genes for methylene tetrahydrofolate reductase (MTHFR) and small nuclear ribonucleoprotein polypeptide N (SNRPN) in infertile males from Romania, in order to establish a correlation with sperm parameters. MTHFR is an enzyme involved in the folate pathway and in de novo nucleotide biosynthesis but also a good example for

  20. 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

  1. 13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

    PubMed Central

    Prabhu, V; Chatson, K B; Abrams, G D; King, J

    1996-01-01

    In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment. PMID:8819325

  2. Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid.

    PubMed

    Buchenau, Bärbel; Thauer, Rudolf K

    2004-10-01

    Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H4F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparent Km below 5 microM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H4F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H4F. From the p-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens.

  3. 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

  4. 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

    2015-10-19

    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.

  5. The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B12- and Tetrahydrofolate-Dependent Chloromethane Metabolism in Methylobacterium extorquens CM4: A Proteomic and Bioinformatics Study

    PubMed Central

    Roselli, Sandro; Nadalig, Thierry; Vuilleumier, Stéphane; Bringel, Françoise

    2013-01-01

    Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate

  6. The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12- and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study.

    PubMed

    Roselli, Sandro; Nadalig, Thierry; Vuilleumier, Stéphane; Bringel, Françoise

    2013-01-01

    Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate

  7. A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6.

    PubMed

    Abe, Tomokuni; Masai, Eiji; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2005-03-01

    Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.

  8. The Inhibition and Resistance Mechanisms of Actinonin, Isolated from Marine Streptomyces sp. NHF165, against Vibrio anguillarum.

    PubMed

    Yang, Na; Sun, Chaomin

    2016-01-01

    Vibrio sp. is the most serious pathogen in marine aquaculture, and the development of anti-Vibrio agents is urgently needed. However, it is extreme lack of high-throughput screening (HTS) model for searching anti-Vibrio compounds. Here, we established a protein-based HTS screening model to identify agents targeting peptide deformylase (PDF) of Vibrio anguillarum. To find potential anti-Vibrio compounds, crude extracts derived from marine actinomycetes were applied for screening with this model. Notably, crude extract of strain Streptomyces sp. NHF165 inhibited dramatically both on V. anguillarum PDF (VaPDF) activity and V. anguillarum cell growth. And actinonin was further identified as the functional component. Anti-VaPDF and anti-V. anguillarum activities of actinonin were dose-dependent, and the IC50 values were 6.94 and 2.85 μM, respectively. To understand the resistance of V. anguillarum against actinonin, spontaneous V. anguillarum mutants with resistance against actinonin were isolated. Surprisingly, for the resistant strains, the region between 774 and 852 base pairs was found to be absent in the gene folD which produces 10-formyl-tetrahydrofolate, a donor of N-formyl to Met-tRNA(fmet). When compared to the wild type strain, ΔfolD mutant showed eight times of minimum inhibition concentration on actinonin, however, the folD complementary strain could not grow on the medium supplemented with actinonin, which suggested that folD gene mutation was mainly responsible for the actinonin resistance. To our knowledge, this is the first report showing that marine derived Streptomyces sp. could produce actinonin with anti-VaPDF activity and the resistance against actinonin by V. anguillarum is mediated by mutation in folD gene. PMID:27679625

  9. The Inhibition and Resistance Mechanisms of Actinonin, Isolated from Marine Streptomyces sp. NHF165, against Vibrio anguillarum

    PubMed Central

    Yang, Na; Sun, Chaomin

    2016-01-01

    Vibrio sp. is the most serious pathogen in marine aquaculture, and the development of anti-Vibrio agents is urgently needed. However, it is extreme lack of high-throughput screening (HTS) model for searching anti-Vibrio compounds. Here, we established a protein-based HTS screening model to identify agents targeting peptide deformylase (PDF) of Vibrio anguillarum. To find potential anti-Vibrio compounds, crude extracts derived from marine actinomycetes were applied for screening with this model. Notably, crude extract of strain Streptomyces sp. NHF165 inhibited dramatically both on V. anguillarum PDF (VaPDF) activity and V. anguillarum cell growth. And actinonin was further identified as the functional component. Anti-VaPDF and anti-V. anguillarum activities of actinonin were dose-dependent, and the IC50 values were 6.94 and 2.85 μM, respectively. To understand the resistance of V. anguillarum against actinonin, spontaneous V. anguillarum mutants with resistance against actinonin were isolated. Surprisingly, for the resistant strains, the region between 774 and 852 base pairs was found to be absent in the gene folD which produces 10-formyl-tetrahydrofolate, a donor of N-formyl to Met-tRNAfmet. When compared to the wild type strain, ΔfolD mutant showed eight times of minimum inhibition concentration on actinonin, however, the folD complementary strain could not grow on the medium supplemented with actinonin, which suggested that folD gene mutation was mainly responsible for the actinonin resistance. To our knowledge, this is the first report showing that marine derived Streptomyces sp. could produce actinonin with anti-VaPDF activity and the resistance against actinonin by V. anguillarum is mediated by mutation in folD gene. PMID:27679625

  10. The Inhibition and Resistance Mechanisms of Actinonin, Isolated from Marine Streptomyces sp. NHF165, against Vibrio anguillarum

    PubMed Central

    Yang, Na; Sun, Chaomin

    2016-01-01

    Vibrio sp. is the most serious pathogen in marine aquaculture, and the development of anti-Vibrio agents is urgently needed. However, it is extreme lack of high-throughput screening (HTS) model for searching anti-Vibrio compounds. Here, we established a protein-based HTS screening model to identify agents targeting peptide deformylase (PDF) of Vibrio anguillarum. To find potential anti-Vibrio compounds, crude extracts derived from marine actinomycetes were applied for screening with this model. Notably, crude extract of strain Streptomyces sp. NHF165 inhibited dramatically both on V. anguillarum PDF (VaPDF) activity and V. anguillarum cell growth. And actinonin was further identified as the functional component. Anti-VaPDF and anti-V. anguillarum activities of actinonin were dose-dependent, and the IC50 values were 6.94 and 2.85 μM, respectively. To understand the resistance of V. anguillarum against actinonin, spontaneous V. anguillarum mutants with resistance against actinonin were isolated. Surprisingly, for the resistant strains, the region between 774 and 852 base pairs was found to be absent in the gene folD which produces 10-formyl-tetrahydrofolate, a donor of N-formyl to Met-tRNAfmet. When compared to the wild type strain, ΔfolD mutant showed eight times of minimum inhibition concentration on actinonin, however, the folD complementary strain could not grow on the medium supplemented with actinonin, which suggested that folD gene mutation was mainly responsible for the actinonin resistance. To our knowledge, this is the first report showing that marine derived Streptomyces sp. could produce actinonin with anti-VaPDF activity and the resistance against actinonin by V. anguillarum is mediated by mutation in folD gene.

  11. 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

  12. Effect of cisplatin on intracellular folate compounds in L1210 cells

    SciTech Connect

    Vitols, K.S.; Monteiano, Y.D.

    1987-05-01

    The biologically active form of the anticancer agent Cisplatin, cis-diamminediaquaplatinum(II)-ion, reacts rapidly with tetrahydrofolate at pH 7 and 37/sup 0/C to form a stable complex. The purified platinum-tetrahydrofolate derivative has also been shown to inhibit the dihydrofolate reductase and the folate transport system of L1210 cells. To determine whether platinum-tetrahydrofolate complex formation would be observed under in vivo conditions, intracellular folates of L1210 cells were labeled by growth on (/sup 3/H)folate and then analyzed by reverse phase HPLC. No evidence for the intracellular formation of platinum tetrahydrofolate was found in cells grown for 48 in the presence of 10/sup -7/ M Cisplatin. The profile of intracellular folate monoglutamates, however, was distinctly different. The level of 5,10-methenyltetrahydrofolate was decreased, and increases were seen in the levels of tetrahydrofolate and its 5-formyl and 10-formyl derivatives. These changes in intracellular folates are compared to those seen when L1210 cells are treated with the antifolate drug, Methotrexate, and the implications for cell kill are examined.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Neonatal hydrocephalus is a result of a block in folate handling and metabolism involving 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Naz, Naila; Jimenez, Alicia Requena; Sanjuan-Vilaplana, Anna; Gurney, Megan; Miyan, Jaleel

    2016-08-01

    Folate is vital in a range of biological processes and folate deficiency is associated with neurodevelopmental disorders such as neural tube defects and hydrocephalus (HC). 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is a key regulator for folate availability and metabolic interconversion for the supply of 1-carbon groups. In previous studies, we found a deficiency of FDH in CSF associated with the developmental deficit in congenital and neonatal HC. In this study, we therefore aimed to investigate the role of FDH in folate transport and metabolism during the brain development of the congenital hydrocephalic Texas (H-Tx) rat and normal (Sprague-Dawley) rats. We show that at embryonic (E) stage E18 and E20, FDH-positive cells and/or vesicles derived from the cortex can bind methyl-folate similarly to folate receptor alpha, the main folate transporter. Hydrocephalic rats expressed diminished nuclear FDH in both liver and brain at all postnatal (P) ages tested (P5, P15, and P20) together with a parallel increase in hepatic nuclear methyl-folate at P5 and cerebral methylfolate at P15 and P20. A similar relationship was found between FDH and 5-methyl cytosine, the main marker for DNA methylation. The data indicated that FDH binds and transports methylfolate in the brain and that decreased liver and brain nuclear expression of FDH is linked with decreased DNA methylation which could be a key factor in the developmental deficits associated with congenital and neonatal HC. Folate deficiency is associated with neurodevelopmental disorders such as neural tube defects and hydrocephalus. 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is a key regulator for folate availability and metabolic interconversion. We show that FDH binds and transports methylfolate in the brain. Moreover, we found that a deficiency of FDH in the nucleus of brain and liver is linked with decreased DNA methylation which could be a key factor in the developmental deficits associated with congenital and

  19. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in chronic myeloid leukemia: an Egyptian study.

    PubMed

    Khorshied, Mervat Mamdooh; Shaheen, Iman Abdel Mohsen; Abu Khalil, Reham E; Sheir, Rania Elsayed

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) gene plays a pivotal role in folate metabolism. Several genetic variations in MTHFR gene as MTHFR-C677T and MTHFR-A1298C result in decreased MTHFR activity, which could influence efficient DNA methylation and explain susceptibility to different cancers. The etiology of chronic myeloid leukemia (CML) is obscure and little is known about individual's susceptibility to CML. In order to assess the influence of these genetic polymorphisms on the susceptibility to CML and its effect on the course of the disease among Egyptians, we performed an age-gender-ethnic matched case-control study. The study included 97 CML patients and 130 healthy controls. Genotyping of MTHFR-C677T and -A1298C was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The results showed no statistical difference in the distribution of MTHFR-C677T and -A1298C polymorphic genotypes between CML patients and controls. The frequency of MTHFR 677-TT homozygous variant was significantly higher in patients with accelerated/blastic transformation phase when compared to those in the chronic phase of the disease. In conclusion, our study revealed that MTHFR-C677T and -A1298C polymorphisms could not be considered as genetic risk factors for CML in Egyptians. However, MTHFR 677-TT homozygous variant might be considered as a molecular predictor for disease progression.

  20. Promiscuous and Adaptable Enzymes Fill “Holes” in the Tetrahydrofolate Pathway in Chlamydia Species

    PubMed Central

    Adams, Nancy E.; Thiaville, Jennifer J.; Proestos, James; Juárez-Vázquez, Ana L.; McCoy, Andrea J.; Barona-Gómez, Francisco; Iwata-Reuyl, Dirk

    2014-01-01

    ABSTRACT Folates are tripartite molecules comprising pterin, para-aminobenzoate (PABA), and glutamate moieties, which are essential cofactors involved in DNA and amino acid synthesis. The obligately intracellular Chlamydia species have lost several biosynthetic pathways for essential nutrients which they can obtain from their host but have retained the capacity to synthesize folate. In most bacteria, synthesis of the pterin moiety of folate requires the FolEQBK enzymes, while synthesis of the PABA moiety is carried out by the PabABC enzymes. Bioinformatic analyses reveal that while members of Chlamydia are missing the genes for FolE (GTP cyclohydrolase) and FolQ, which catalyze the initial steps in de novo synthesis of the pterin moiety, they have genes for the rest of the pterin pathway. We screened a chlamydial genomic library in deletion mutants of Escherichia coli to identify the “missing genes” and identified a novel enzyme, TrpFCtL2, which has broad substrate specificity. TrpFCtL2, in combination with GTP cyclohydrolase II (RibA), the first enzyme of riboflavin synthesis, provides a bypass of the first two canonical steps in folate synthesis catalyzed by FolE and FolQ. Notably, TrpFCtL2 retains the phosphoribosyl anthranilate isomerase activity of the original annotation. Additionally, we independently confirmed the recent discovery of a novel enzyme, CT610, which uses an unknown precursor to synthesize PABA and complements E. coli mutants with deletions of pabA, pabB, or pabC. Thus, Chlamydia species have evolved a variant folate synthesis pathway that employs a patchwork of promiscuous and adaptable enzymes recruited from other biosynthetic pathways. PMID:25006229

  1. 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

  2. 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.

  3. Retinal Ganglion Cell Loss and Mild Vasculopathy in Methylene Tetrahydrofolate Reductase (Mthfr)-Deficient Mice: A Model of Mild Hyperhomocysteinemia

    PubMed Central

    Markand, Shanu; Saul, Alan; Roon, Penny; Prasad, Puttur; Martin, Pamela; Rozen, Rima; Ganapathy, Vadivel; Smith, Sylvia B.

    2015-01-01

    Purpose. Methylenetetrahydrofolate reductase (Mthfr) is a key enzyme in homocysteine-methionine metabolism. We investigated Mthfr expression in retina and asked whether mild hyperhomocysteinemia, due to Mthfr deficiency, alters retinal neurovascular structure and function. Methods. Expression of Mthfr was investigated at the gene and protein level using quantitative (q) RT-PCR, in situ hybridization, immunoblotting, and immunohistochemistry (IHC). The Mthfr+/+ and Mthfr+/− mice were subjected to comprehensive evaluation using ERG, funduscopy, fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), HPLC, and morphometric and IHC analysis of glial fibrillary acidic protein (GFAP) at 8 to 24 weeks. Results. Gene and protein analyses disclosed widespread retinal expression of Mthfr. Electroretinography (ERG) revealed a significant decrease in positive scotopic threshold response in retinas of Mthfr+/− mice at 24 weeks. Fundus examination in mice from both groups was normal; FA revealed areas of focal vascular leakage in 20% of Mthfr+/− mice at 12 to 16 weeks and 60% by 24 weeks. The SD-OCT revealed a significant decrease in nerve fiber layer (NFL) thickness at 24 weeks in Mthfr+/− compared to Mthfr+/+ mice. There was a 2-fold elevation in retinal hcy at 24 weeks in Mthfr+/− mice by HPLC and IHC. Morphometric analysis revealed an approximately 20% reduction in cells in the ganglion cell layer of Mthfr+/− mice at 24 weeks. The IHC indicated significantly increased GFAP labeling suggestive of Müller cell activation. Conclusions. Mildly hyperhomocysteinemic Mthfr+/− mice demonstrate reduced ganglion cell function, thinner NFL, and mild vasculopathy by 24 weeks. The retinal phenotype is similar to that of hyperhomocysteinemic mice with deficiency of cystathionine-β-synthase (Cbs) reported earlier. The data support the hypothesis that hyperhomocysteinemia may be causative in certain retinal neurovasculopathies. PMID:25766590

  4. 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

  5. [Intake of folic acid in the total daily diet--effect of food preparation on its folic acid content].

    PubMed

    Müller, H

    1995-03-01

    The folic acid content of total daily diet was determined by means of high-performance liquid chromatography (HPLC). The contents of tetrahydrofolic acid (THF), 5-methyl-THF and 5-formyl-THF were differentiated. The mean of the folic acid content of the total daily diet samples determined analytically was 205 +/- 60 micrograms and the mean of the individual ingredients of the samples was 401 +/- 78 micrograms, which implies that about 50% of folic acid is destroyed by common household food preparation methods. If the contents of pteroylglutamic acid (PteGlu) and 10-formyl-PteGlu (which cannot be determined analytically) are added, it can be assumed that the folic acid content with only be reduced by about 40%. THF and 5-methyl-THF proved to be less stable than 5-formyl-THF. The monoglutamate portion of the total folat content was higher in the total diet samples than in the individual foodstuffs as a consequence of the action of the enzyme "deconjugase" which is released when the matrix of food-stuffs is destroyed.

  6. Formate: The Neglected Member of One-Carbon Metabolism.

    PubMed

    Brosnan, Margaret E; Brosnan, John T

    2016-07-17

    Formate, the only non-tetrahydrofolate (THF)-linked intermediate in one-carbon metabolism, is produced in mammals from a variety of metabolic sources. It occurs in serum of adults at a concentration of approximately 30 μM. Its principal function lies as a source of one-carbon groups for the synthesis of 10-formyl-THF and other one-carbon intermediates; these are primarily used for purine synthesis, thymidylate synthesis, and the provision of methyl groups for synthetic, regulatory, and epigenetic methylation reactions. Although formate is largely produced in mitochondria, these functions mostly occur in the cytoplasm and nucleus. Formate plays a significant role in embryonic development, as evidenced by the effectiveness of formate in the pregnant dam's drinking water on the incidence of neural tube defects in some genetic models. High formate concentrations in fetal lambs may indicate a role in fetal development and suggest that extracellular formate may play a role in the interorgan distribution of one-carbon groups.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Folate-Dependent Purine Nucleotide Biosynthesis in Humans1

    PubMed Central

    Baggott, Joseph E; Tamura, Tsunenobu

    2015-01-01

    Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases—5′-phosphoribosyl-glycinamide (GAR) and 5′-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases—to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease. PMID:26374178

  13. Folate-Dependent Purine Nucleotide Biosynthesis in Humans.

    PubMed

    Baggott, Joseph E; Tamura, Tsunenobu

    2015-09-01

    Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease. PMID:26374178

  14. 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...

  15. Activity of formylphosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

    Jahansouz, H.; Kofron, J.L.; Smithers, G.W.; Himes, R.H.; Reed, G.H.

    1986-05-01

    Formylphosphate (FP), a putative enzyme-bound intermediate in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase, was synthesized from formylfluoride and Pi. Measurement of hydrolysis rates by /sup 31/P NMR showed that FP is very unstable with a half-life of 48 min at 20/sup 0/C and pH 7. At pH 7 hydrolysis occurs with O-P bond cleavage as shown by /sup 18/O incorporation from /sup 18/O-H/sub 2/O into Pi. The substrate activity of FP was tested in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase isolated from Clostridium cylindrosporum. MgATP + H/sub 4/folate + HCOO/sup -/ in equilibrium MgADP + Pi +N/sup 10/-formylH/sub 4/folate FP supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formylH/sub 4/folate is produced from H/sub 4/-folate and FP but only if ADP is present, and ATP is produced from FP and ADP but only if H/sub 4/folate is present. The requirements for H/sub 4/folate in the synthesis of ATP from ADP and FP and for ADP in the synthesis of N/sup 10/-formylH/sub 4/folate from FP and H/sub 4/folate, are consistent with past kinetic and isotope exchange studies which showed that the reaction proceeds by a sequential mechanism and that all three substrates must be present for any reaction to occur.

  16. Channelling and formation of ‘active’ formaldehyde in dimethylglycine oxidase

    PubMed Central

    Leys, David; Basran, Jaswir; Scrutton, Nigel S.

    2003-01-01

    Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes dimethylglycine to a labile iminium intermediate. The C-terminal region binds tetrahydrofolate, comprises three domains arranged in a ring-like structure and is related to the T-protein of the glycine cleavage system. The complex with folinic acid indicates that this enzyme selectively activates the N10 amino group for initial attack on the substrate. Dead-end reactions with oxidized folate are avoided by the strict stereochemical constraints imposed by the folate-binding funnel. The active sites in DMGO are ∼40 Å apart, connected by a large irregular internal cavity. The tetrahydrofolate-binding funnel serves as a transient entry–exit port, and access to the internal cavity is controlled kinetically by tetrahydrofolate binding. The internal cavity enables sequestration of the reactive iminium intermediate prior to reaction with tetrahydrofolate and avoids formation of toxic formaldehyde. This mode of channelling in DMGO is distinct from other channelling mechanisms. PMID:12912903

  17. 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

  18. 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

  19. 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 ...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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…

  5. 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...

  6. Biosynthesis of purines by a proplastid fraction from soybean nodules.

    PubMed

    Boland, M J; Schubert, K R

    1983-01-01

    A proplastid-containing fraction was rapidly prepared from soybean nodules by a combination of differential and step gradient centrifugation. This fraction was capable of incorporating [U-14C]glycine into purines in the presence of added phosphoribosylpyrophosphate or ribose 5-phosphate, glutamine, aspartate, ATP, bicarbonate, methenyl tetrahydrofolate, MgCl2, and KCl. The primary product was IMP; some inosine was also formed. Soluble and bacteroid fractions from soybean nodules gave considerably lower rates of incorporation. Labeled carbon from both [U-14C]serine and [3-14C]serine was incorporated into purines when tetrahydrofolate and NADP+ were substituted for methenyl tetrahydrofolate. In this case, small amounts of label were also found in AMP and xanthine monophosphate (XMP). Labeled bicarbonate was incorporated into IMP and inosine by the proplastid fraction. Labeled formate, however, was not a competent substrate for purine synthesis, indicating the absence of formyl tetrahydrofolate synthetase activity in this fraction. When labeled IMP was incubated with a proplastid preparation, most of the label appeared in inosine. XMP and xanthosine were also formed if NAD+ or NADP+ was added to the incubation mixture indicating the presence of IMP dehydrogenase activity in the proplastid fraction.

  7. Neither folic acid supplementation nor pregnancy affects the distribution of folate forms in the red blood cells of women.

    PubMed

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

    2014-09-01

    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 forms did

  8. [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.

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

    PubMed Central

    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

  10. 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

  11. O-demethylase from Acetobacterium dehalogenans--cloning, sequencing, and active expression of the gene encoding the corrinoid protein.

    PubMed

    Kaufmann, F; Wohlfarth, G; Diekert, G

    1998-10-15

    The ether-cleaving O-demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4-hydroxy-3-methoxy-benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25-kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein:tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino-terminal sequences of the corrinoid protein and the vanillate:corrinoid protein methyltransferase (methyltransferase I), a 723-bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O-demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60% similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen-insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O-demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltranSferase II

  12. 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

  13. Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

    PubMed Central

    Delle Fratte, S; White, R H; Maras, B; Bossa, F; Schirch, V

    1997-01-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens. PMID:9393711

  14. 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.

  15. O-demethylase from Acetobacterium dehalogenans--substrate specificity and function of the participating proteins.

    PubMed

    Kaufmann, F; Wohlfarth, G; Diekert, G

    1998-05-01

    The ether-cleaving O-demethylase isolated from syringate-grown cells of Acetobacterium dehalogenans (formerly named strain MC) consists of four proteins, components A, B, C and D. The enzyme system converts only phenyl methyl ethers with a hydroxyl group in the ortho position to the methoxyl moiety. The presence of a carboxyl group in the aromatic compound was not required for O-demethylase reaction. Component B mediated the conversion of vanillate to 3,4-dihydroxybenzoate in the presence of the Ti(III)-reduced corrinoid-containing component A. After addition of component D and tetrahydrofolate, methyl tetrahydrofolate was formed from vanillate in stoichiometric amounts. Titanium(III) citrate as a reductant could be replaced by H2, methyl viologen or ferredoxin, partially purified hydrogenase, purified component C obtained from A. dehalogenans, and ATP. From these findings, it was deduced that component B serves as vanillate:corrinoid protein methyltransferase (methyltransferase I) mediating the methyl transfer from vanillate to the reduced corrinoid protein component A. Component D functions as methylcorrinoid protein:tetrahydrofolate transferase (methyltransferase II). The role of component C is probably that of an activating protein reversing accidental oxidation of the protein-bound cob(I)alamin to cob(II)alamin in the presence of ATP and reducing equivalents supplied by the enzymatic oxidation of hydrogen.

  16. Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids.

    PubMed

    Wang, E A; Kallen, R; Walsh, C

    1981-07-10

    Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.

  17. Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

    PubMed Central

    Lamarre, Simon G.; Molloy, Anne M.; Reinke, Stacey N.; Sykes, Brian D.; Brosnan, Margaret E.

    2012-01-01

    Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. Am J Physiol Endocrinol Metab 301: E000–E000, 2011. First published September 20, 2011; 10.1152/ajpendo.00345.2011.—We carried out a 1H-NMR metabolomic analysis of sera from vitamin B12-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 μM in control rats to 402 μM in vitamin B12-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B12- and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B6-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine β-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways. PMID:21934042

  18. 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.

  19. Identification of an H2-M3-Restricted Listeria Epitope: Implications for Antigen Presentation by M3

    PubMed Central

    Lenz, Laurel L.; Dere, Beverley; Bevan, Michael J.

    2009-01-01

    Summary Using expression cloning, we have identified an H2-M3-restricted epitope of the intracellular bacterial pathogen Listeria monocytogenes. Picomolar concentrations of an amino-terminal N-formylated hexapeptide, fMIGWII, targeted cells for lysis by CD8+ cytotoxic T cells, while the nonformylated peptide was approximately 100-fold less active. The sequence of the 185 aa protein source of this epitope predicts a transmembrane protein that retains its N terminus and assumes an Nout–Cin topology. This membrane orientation offers an explanation for the protection of the epitope from deformylases present in the bacterial cell and suggests an explanation for the ability of phagocytes to present H2-M3-restricted bacterial epitopes via a vacuolar TAP-independent mechanism. PMID:8758895

  20. 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.

  1. Novel Isonitrile Hydratase Involved in Isonitrile Metabolism*

    PubMed Central

    Sato, Hiroyoshi; Hashimoto, Yoshiteru; Fukatsu, Hiroshi; Kobayashi, Michihiko

    2010-01-01

    We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., and Kobayashi, M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13726–13731). In this study, we found an enzyme involved in the first step of isonitrile metabolism, isonitrile hydratase, that hydrates isonitrile to the corresponding N-substituted formamide. First, we investigated the optimum culture conditions for the production of isonitrile hydratase. The highest enzyme activity was obtained when A. pascens F164 was cultured in a nutrient medium containing N-benzylformamide. This Arthrobacter isonitrile hydratase was purified, characterized, and compared with Pseudomonas putida N19-2 isonitrile hydratase (InhA), which is the sole one reported at present. Arthrobacter isonitrile hydratase was found to have a molecular mass of about 530 kDa and to consist of 12 identical subunits. The apparent Km value for cyclohexyl isocyanide was 0.95 ± 0.05 mm. A. pascens F164 grew and exhibited the isonitrile hydratase and N-substituted formamide deformylase activities when cultured in a medium containing an isonitrile as the sole carbon and nitrogen sources. However, both enzyme activities were not observed on culture in a medium containing glycerol and (NH4)2SO4 as the sole carbon and nitrogen sources, respectively. These findings suggested that the Arthrobacter enzyme is an inducible enzyme, possibly involved in assimilation and/or detoxification of isonitrile. Moreover, gene cloning of the Arthrobacter enzyme revealed no sequence similarity between this enzyme and InhA. Comparison of their properties and features demonstrated that the two enzymes are biochemically, immunologically, and structurally different from each other. Thus, we discovered a new isonitrile hydratase named InhB. PMID:20826798

  2. 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

  3. 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

  4. Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

    Smithers, G.W.; Jahansouz, H.; Kofron, J.L.; Himes, R.H.; Reed, G.H.

    1987-06-30

    Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate, and the product was characterized by /sup 31/P, /sup 1/H, and /sup 13/C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by /sup 31/P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 /sup 0/C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by /sup 18/O incorporation from H/sub 2//sup 18/O into P/sub i/, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k/sub cat/ values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.

  5. Mammalian folylpoly-. gamma. -glutamate synthetase. 2. Substrate specificity and kinetic properties

    SciTech Connect

    Cichowicz, D.J.; Shane, B.

    1987-01-27

    The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and L-(/sup 14/C)glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis while 5- and 10-position substitutions of the folate molecule impair catalysis. k/sub cat/ values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The K/sub m/ for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and ..beta..,..gamma..-methylene-ATP, ..beta..,..gamma..-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P/sup 1/,P/sup 5/-di(adenosine-5') pentaphosphate, and free ATP/sup 4 -/ are potent inhibitors of the reaction.

  6. 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.

  7. [Vitamin B9].

    PubMed

    Guilland, Jean-Claude; Aimone-Gastin, Isabelle

    2013-10-01

    Vitamin B9 is represented by the group of folate, whose structure is derived from folic acid. The biologically active form is reduced tetrahydrofolates, serving as an essential cofactor in methylation reactions, including the vitamin B12-dependent formation of methionine from homocysteine, and as a carrier of one-carbon units involved in the synthesis of purines and pyrimidines. Folate deficiency is associated with hyperhomocysteinemia, megaloblastic anemia, leuco- and thrombocytopenia, cardiovascular disease, embryonic defects, in particular neural tube defects, and, possibly, malignancies, depression and cognitive impairment. PMID:24298825

  8. 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.

  9. 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

  10. 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

  11. Serine hydroxymethyltransferase 1 and 2: gene sequence variation and functional genomic characterization.

    PubMed

    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

    2012-03-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a β-carbon from serine to tetrahydrofolate to form glycine and 5,10-methylene-tetrahydrofolate. 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 non-synonymous single-nucleotide polymorphism (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<1.0E-10) with SHMT1 messenger RNA 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 messenger RNA 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.

  12. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

    SciTech Connect

    Kamen, B.A.; Wang, M.T.; Streckfuss, A.J.; Peryea, X.; Anderson, R.G.

    1988-09-25

    MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl(/sup 3/H)tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl(/sup 3/H)tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface.

  13. Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

    SciTech Connect

    Elliott, J.I.; Ljungdahl, L.G.

    1982-04-01

    The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4'-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K/sub m/ values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4'-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20 to 30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20 to 30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.

  14. Folates as adjuvants to anticancer agents: Chemical rationale and mechanism of action.

    PubMed

    Danenberg, Peter V; Gustavsson, Bengt; Johnston, Patrick; Lindberg, Per; Moser, Rudolf; Odin, Elisabeth; Peters, Godefridus J; Petrelli, Nicholas

    2016-10-01

    Folates have been used with cytotoxic agents for decades and today they are used in hundreds of thousands of patients annually. Folate metabolism is complex. In the treatment of cancer with 5-fluorouracil, the administration of folates mechanistically leads to the formation of [6R]-5,10-methylene-tetrahydrofolate, and the increased concentration of this molecule leads to stabilization of the ternary complex comprising thymidylate synthase, 2'-deoxy-uridine-5'-monophosphate, and [6R]-5,10-methylene-tetrahydrofolate. The latter is the only natural folate that can bind directly in the ternary complex, with other folates requiring metabolic activation. Modulation of thymidylate synthase activity became central in the study of folate/cytotoxic combinations and, despite wide use, research into the folate component was neglected, leaving important questions unanswered. This article revisits the mechanisms of action of folates and evaluates commercially available folate derivatives in the light of current research. Better genomic insight and availability of new analytical techniques and stable folate compounds may open new avenues of research and therapy, ultimately bringing increased clinical benefit to patients. PMID:27637357

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Effect of folate derivatives on the activity of antifolate drugs used against malaria and cancer

    PubMed Central

    Nduati, Eunice; Diriye, Abdi; Ommeh, Sheila; Mwai, Leah; Kiara, Steven; Masseno, Victor; Kokwaro, Gilbert

    2008-01-01

    The folate derivatives folic acid (FA) and folinic acid (FNA) decrease the in vivo and in vitro activities of antifolate drugs in Plasmodium falciparum. However, the effects of 5-methyl-tetrahydrofolate (5-Me-THF) and tetrahydrofolate (THF), the two dominant circulating folate forms in humans, have not been explored yet. We have investigated the effects of FA, FNA, 5-Me-THF, and THF on the in vitro activity of the antimalarial antifolates pyrimethamine and chlorcycloguanil and the anticancer antifolates methotrexate (MTX), aminopterin, and trimetrexate (TMX), against P. falciparum. The results indicate that these anticancers are potent against P. falciparum, with IC50 < 50 nM. 5-Me-THF does not significantly decrease the activity of all tested drugs, and none of the tested folate derivatives significantly decrease the activity of these anticancers. Thus, malaria folate metabolism has features different from those in human, and the exploitation of this difference could lead to the discovery of new drugs to treat malaria. For instance, the combination of 5-Me-THF with a low dose of TMX could be used to treat malaria. In addition, the safety of a low dose of MTX in the treatment of arthritis indicates that this drug could be used alone to treat malaria. Electronic supplementary material The online version of this article (doi:10.1007/s00436-008-0897-4) contains supplementary material, which is available to authorized users. PMID:18259776

  20. 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.

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

    PubMed Central

    Cufí, Sílvia; Martin-Castillo, Begoña; Micol, Vicente; Joven, Jorge; Segura-Carretero, Antonio; Menendez, Javier A.

    2012-01-01

    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

  3. [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

  4. [The role of homocysteine and methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase polymorphisms in the development of cardiovascular diseases and hypertension].

    PubMed

    Marosi, Krisztina; Agota, Annamária; Végh, Veronika; Joó, József Gábor; Langmár, Zoltán; Kriszbacher, Ildikó; Nagy, Zsolt B

    2012-03-25

    Cardiovascular diseases (CVDs) are the leading causes of death in the developed countries. Elevated homocysteine level is as an independent risk factor of CVDs. The C677T and A1298C variants of methylenetetrahydrofolate reductase gene (MTHFR) have been shown to influence folate and homocysteine metabolisms. However, the relationship between MTHFR polymorphisms and hyperhomocysteinemia has not been well established yet. The gene variants were also reported to be associated with CVDs. In addition, the C677T polymorphisms may play a role in the development of hypertension. Recent research evidence has suggested that MTHFR variants might be independently linked to CVDs and hypertension, because of the involvement of the MTHFR enzyme product (5-methyl-tetrahydrofolate /5-MTHF) in the regulation of endothelial functions. Further research is required to investigate the association between gene polymorphisms of folate-metabolizing enzymes and CVDs, and to identify the possible role of the relevant gene variants in the molecular pathogenesis of hyperhomocysteinemia.

  5. [Neonatal renal vein thrombosis in a heterozygous carrier of both factor V Leiden and the MTHFR gene mutation].

    PubMed

    Wannes, S; Soua, H; Ghanmi, S; Braham, H; Hassine, M; Hamza, H A; Ben Hamouda, H; Sfar, M-T

    2012-04-01

    Renal vein thrombosis (RVT) is a rare but potentially serious neonatal disease. Its epidemiology and its clinical and biological expression are currently well known, but its etiological exploration, like that of venous thromboembolism, is increasingly complex. Perinatal risk factors such as prematurity, dehydration, and birth asphyxia have lost their direct accountability at the expense of their interaction with constitutional disorders of hemostasis. We report a case of RVT in a newborn who was a heterozygous carrier of both factor V Leiden and the methylene tetrahydrofolate reductase (MTHFR) gene mutation. We recall the clinical and epidemiological characteristics. A search for inborn blood coagulation disorders should be systematic in the newborn infant with venous thrombosis because of the risk of recurrence, taking into account perinatal factors and maternal thrombophilia (especially if RVT is established during the prenatal period).

  6. [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.

  7. Increased neurotoxicity of arsenic in methylenetetrahydrofolate reductase deficiency.

    PubMed

    Brouwer, O F; Onkenhout, W; Edelbroek, P M; de Kom, J F; de Wolff, F A; Peters, A C

    1992-01-01

    A 16-year-old girl from Surinam presented with mental deterioration and severe paraparesis with areflexia and bilateral Babinski signs. Laboratory examination showed a hyperhomocysteinemia that was caused by 5,10-methylene-tetrahydrofolate reductase (MTHFR) deficiency. In addition, urine samples contained large amounts of arsenic. An open bag with the pesticide copper acetate arsenite was found to be the source of exposure. In remethylation defects such as MTHFR deficiency, the concentration of methyldonors is severely reduced. As arsenic is detoxified by methylation, we suggest that the MTHFR deficiency in this girl might explain the fact that of all family members exposed to arsenic, only she developed severe clinical signs and symptoms of arsenic poisoning.

  8. FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

    PubMed Central

    Gabrielsen, Mads; Beckham, Katherine S.H.; Cogdell, Richard J.; Byron, Olwyn; Roe, Andrew J.

    2012-01-01

    FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution. Structured summary of protein interactions FolX and FolXbind by x-ray crystallography (View interaction) FolX and FolXbind by cosedimentation in solution (View interaction) FolX and FolXbind by x ray scattering (View interaction) PMID:22575651

  9. 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 PAGES

    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

  10. 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.

  11. Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N.

    PubMed

    Ganji, Roopa Jones; Reddi, Ravikumar; Gumpena, Rajesh; Marapaka, Anil Kumar; Arya, Tarun; Sankoju, Priyanka; Bhukya, Supriya; Addlagatta, Anthony

    2015-05-01

    Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

  12. 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

  13. 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

  14. 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.

  15. Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N

    PubMed Central

    Ganji, Roopa Jones; Reddi, Ravikumar; Gumpena, Rajesh; Marapaka, Anil Kumar; Arya, Tarun; Sankoju, Priyanka; Bhukya, Supriya; Addlagatta, Anthony

    2015-01-01

    Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases. PMID:25644575

  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

    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

    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.

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

    PubMed

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

    2014-10-28

    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.

  20. 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

  1. 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

  2. Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6.

    PubMed

    Kasai, Daisuke; Masai, Eiji; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2004-08-01

    Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the beta subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3MGA O-demethylase, and gallate dioxygenase are participants.

  3. Substrate specificity of mammalian folylpolyglutamate synthetase for 5,10-dideazatetrahydrofolate analogs.

    PubMed

    Habeck, L L; Mendelsohn, L G; Shih, C; Taylor, E C; Colman, P D; Gossett, L S; Leitner, T A; Schultz, R M; Andis, S L; Moran, R G

    1995-08-01

    The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.

  4. Genome Sequences of Two Closely Related Vibrio parahaemolyticus Phages, VP16T and VP16C†

    PubMed Central

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

    2003-01-01

    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

  5. Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440.

    PubMed

    Jiménez, José I; Canales, Angeles; Jiménez-Barbero, Jesús; Ginalski, Krzysztof; Rychlewski, Leszek; García, José L; Díaz, Eduardo

    2008-08-12

    The aerobic catabolism of nicotinic acid (NA) is considered a model system for degradation of N-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural-functional relationships of most of the enzymes involved in this process are still unknown. We have characterized a gene cluster (nic genes) from Pseudomonas putida KT2440 responsible for the aerobic NA degradation in this bacterium and when expressed in heterologous hosts. The biochemistry of the NA degradation through the formation of 2,5-dihydroxypyridine and maleamic acid has been revisited, and some gene products become the prototype of new types of enzymes with unprecedented molecular architectures. Thus, the initial hydroxylation of NA is catalyzed by a two-component hydroxylase (NicAB) that constitutes the first member of the xanthine dehydrogenase family whose electron transport chain to molecular oxygen includes a cytochrome c domain. The Fe(2+)-dependent dioxygenase (NicX) converts 2,5-dihydroxypyridine into N-formylmaleamic acid, and it becomes the founding member of a new family of extradiol ring-cleavage dioxygenases. Further conversion of N-formylmaleamic acid to formic and maleamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some members of the alpha/beta-hydrolase fold superfamily. This work allows exploration of the existence of orthologous gene clusters in saprophytic bacteria and some pathogens, where they might stimulate studies on their role in virulence, and it provides a framework to develop new biotechnological processes for detoxification/biotransformation of N-heterocyclic aromatic compounds.

  6. Safety, tolerability, and efficacy of GSK1322322 in the treatment of acute bacterial skin and skin structure infections.

    PubMed

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

    2014-11-01

    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

  7. 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

  8. Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

    2005-08-01

    Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

  9. 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.

  10. 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

    2016-04-01

    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.

  11. 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

  12. A fast chemo-enzymatic synthesis of [11C]-N5,N10-methylenetetrahydrofolate as a potential PET tracer for proliferating cells☆

    PubMed Central

    Saeed, Muhammad; Tewson, Timothy J.; Erdahl, Colbin E.; Kohen, Amnon

    2015-01-01

    Introduction Thymidylate synthase (TSase) and folate receptors (FRs) are well developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of 11CH3I to [11C]-formaldehyde (11CH2O) encouraged us to label the co-factor of this enzyme. Preliminary studies conducted on cell lines have demonstrated a preferential uptake of [11-14C]-(R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (14CH2H4folate) by cancerous cell vs. normal cells from the same organ [1] pointing out 11CH2H4folate as a PET tracer for cancer imaging. Herein we report the synthesis of 11CH2H4folate, which may serve as a potential positron emission tomography (PET) tracer. Methods In a remotely controlled module, 11CH3I was bubbled into a reaction vial containing trimethylamine N-oxide (TMAO) in DMF and heated to 70 °C for 2 min. 11CH2O formed after the completion of reaction was then mixed with a solution of freshly prepared tetrahydrofolate (H4folate) by using a fast chemo-enzymatic approach to accomplish synthesis of 11CH2H4folate. Purification of the product was carried out by loading the crude reaction mixture on a SAX cartridge, washing with water to remove unbound impurities and finally eluting with a saline solution. Results The synthesis and purification of 11CH2H4folate was completed within 5 min. HPLC analysis of the product after SAX purification indicate more than 90% of the radioactivity that was retained on the SAX cartridge was in 11CH2H4folate with minor (<10%) radioactivity due to unreacted 11CH2O. Conclusion We present a fast (~5 min) synthesis and purification of 11CH2H4folate, as a potential PET tracer. The final product is received in physiological compatible buffer (100 mM sodium phosphate, pH 7.0 containing 500 mM NaCl) and ready for use in vivo. PMID:22300960

  13. [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

  14. 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

  15. 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.

  16. Folate and homocysteine metabolisms and their roles in the biochemical basis of neuropsychiatry.

    PubMed

    Coşar, Alpaslan; Ipçioğlu, Osman Metin; Ozcan, Omer; Gültepe, Mustafa

    2014-01-01

    The term 'one-carbon metabolism' is commonly used to describe 3 separate metabolic processes: folate metabolism, the homocysteine remethylation cycle, and the transsulfuration pathway. Folate metabolism concerns the biochemical reactions in which endogenous and exogenous one-carbon units are transferred to tetrahydrofolates. The remethylation cycle is used for the synthesis of methionine from homocysteine with one-carbon units that come from folate. This methionine is thenfused for the synthesis of S-adenosyl methionine, which is a general donor of methyl groups for many biochemical reactions in the human body. In the transsulfuration pathway, some amino acids and polypeptides, such as cystathionine, cysteine, and glutathione, are synthesized from homocysteine. The kinetics of the enzymes in this pathway are regulated by the substrates of the remethylation cycle. The methylation process has been thought to have an important role in the biochemical basis of neuropsychiatry. An elevated homocysteine level is the most important marker of folate and vitamin B12 deficiencies, and also the most reliable biochemical sign of functional insufficiency. Some neurological and neuropsychiatric diseases, such as psychosis, Alzheimer's disease, and autism, have been found to be related to disorders of one-carbon metabolism. This review aims to summarize both one-carbon metabolism and its relationships with neuropsychiatric disorders.

  17. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis.

    PubMed

    Fang, Jing Yuan; Xiao, Shu Dong

    2003-01-01

    DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, A-->G at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.

  18. 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

  19. Epigenetic regulation of aortic remodeling in hyperhomocysteinemia

    PubMed Central

    Narayanan, Nithya; Pushpakumar, Sathnur Basappa; Givvimani, Srikanth; Kundu, Sourav; Metreveli, Naira; James, Dexter; Bratcher, Adrienne P.; Tyagi, Suresh C.

    2014-01-01

    Hyperhomocysteinemia (HHcy) is prevalent in patients with hypertension and is an independent risk factor for aortic pathologies. HHcy is known to cause an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to the accumulation of collagen in the aorta and resulting in stiffness and development of hypertension. Although the exact mechanism of extracellular matrix (ECM) remodeling is unclear, emerging evidence implicates epigenetic regulation involving DNA methylation. Our purpose was to investigate whether 5-aza-2′-deoxycytidine (Aza), a DNA methyltransferase (DNMT1) inhibitor, reduces high blood pressure (BP) by regulating aortic ECM remodeling in HHcy. Wild-type and cystathionine β-synthase (CBS)+/− HHcy mice were treated with Aza (0.5 mg/kg body weight). In HHcy mice, Aza treatment normalized the plasma homocysteine (Hcy) level and BP. Thoracic and abdominal aorta ultrasound revealed a reduction in the resistive index and wall-to-lumen ratio. Vascular response to phenylephrine, acetylcholine, and sodium nitroprusside improved after Aza in HHcy mice. Histology showed a marked reduction in collagen deposition in the aorta. Aza treatment decreased the expression of DNMT1, MMP9, TIMP1, and S-adenosyl homocysteine hydrolase (SAHH) and upregulated methylene tetrahydrofolate reductase (MTHFR). We conclude that reduction of DNA methylation by Aza in HHcy reduces adverse aortic remodeling to mitigate hypertension.—Narayanan, N., Pushpakumar, S. B., Givvimani, S., Kundu, S., Metreveli, N., James, D., Bratcher, A. P., Tyagi, S. C. Epigenetic regulation of aortic remodeling in hyperhomocysteinemia. PMID:24739303

  20. Methionine, folic acid and vitamin B12 in growing-finishing pigs: impact on growth performance and meat quality.

    PubMed

    Giguére, Alain; Girard, Christiane L; Matte, J Jacques

    2008-06-01

    Growth performance, metabolic variables, and meat quality were measured in 78 growing-finishing pigs using supplements of 0 (C), or 0.2% of DL-methionine (M), and three combinations of folic acid [mg/kg] and cyanocobalamin [microg/kg], respectively 0 and 0 (V0), 10 and 25 (V1), and 10 and 150 (V2) in a 2 x 3 factorial arrangement. Feed conversion was lower (p = 0.05) in M than in C pigs during the growing period (0-4 weeks). Both V1 and V2 treatments increased plasma vitamin B12 (p < 0.01) and decreased plasma homocysteine (p < 0.01). Plasma 5-methyl-tetrahydrofolates were the lowest, highest and intermediate in V0, V1 and V2 pigs (p < 0.04), respectively. In V2 meat, folates were 32% higher, vitamin B12, 55% higher and homocysteine, 28% lower than in V0 (p < 0.01). Oxidative stability of the fresh meat was similar among treatments during a storage period of 42 days. Therefore, methionine supplements improved growth performance during the growing period. Vitamin supplements interacted with the methionine cycle pathway, increased vitamin content of pork meat but did not improve oxidative stability of the fresh meat during storage.

  1. Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part I--Folate, Vitamin B12, Vitamin B6.

    PubMed

    Simpson, Joe Leigh; Bailey, Lynn B; Pietrzik, Klaus; Shane, Barry; Holzgreve, Wolfgang

    2010-12-01

    This two-part review highlights micronutrients for which either public health policy has been established or for which new evidence provides guidance as to recommended intakes during pregnancy. One pivotal micronutrient is folate, the generic name for different forms of a water-soluble vitamin essential for the synthesis of thymidylate and purines and, hence, DNA. For non-pregnant adult women the recommended intake is 400 μg/day dietary folate equivalent. For women capable of becoming pregnant an additional 400 μg/day of synthetic folic acid from supplements or fortified foods is recommended to reduce the risk of neural tube defects (NTD). The average amount of folic acid received through food fortification (grains) in the US is only 128 μg/day, emphasising the need for the supplemental vitamin for women of reproductive age. Vitamin B12 (cobalamin) is a cofactor required for enzyme reactions, including generation of methionine and tetrahydrofolate. B12 is found almost exclusively in foods of animal origin (meats, dairy products); therefore, vegetarians are at greatest risk for dietary vitamin B12 deficiency and should be supplemented. Vitamin B6 is required for many reactions, primarily in amino acid metabolism. Meat, fish and poultry are good dietary sources. Supplementation beyond routine prenatal vitamins is not recommended.

  2. 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.

  3. Addressing a folate imbalance in fetal cerebrospinal fluid can decrease the incidence of congenital hydrocephalus.

    PubMed

    Cains, Sarah; Shepherd, Andrew; Nabiuni, Mohammad; Owen-Lynch, Penelope Jane; Miyan, Jaleel

    2009-04-01

    Fetal-onset hydrocephalus (HC), which affects between 1:500 and 1:5000 live human births, results from unequal production and drainage of cerebrospinal fluid (CSF) and is associated with abnormal development of the cerebral cortex leading to severe neurological deficits. We previously found that in the hydrocephalic Texas rat, the CSF of affected fetuses induced a cell cycle arrest in neural progenitor cells. Here, we show that alterations in folate metabolism in the CSF of the developing cerebrum are likely responsible for this effect. We identified 3 folate enzymes in the CSF and demonstrate that low levels of one of these, 10-formyltetrahydrofolate dehydrogenase, are associated with HC in the hydrocephalic Texas rat. Therefore, we tested whether supplementation with specific folate species would improve developmental outcome. After daily administration of a combination of tetrahydrofolic and 5-formyltetrahydrofolic acids to pregnant dams, there was a significant reduction in the incidence of HC and improved brain development. By contrast, supplementation with folic acid increased the incidence of congenital HC in this model. These results indicate the complexities of folate metabolism in the developing brain and suggest that folate imbalance leading to HC in the hydrocephalic Texas rat fetuses can be treated with maternal folate supplementation using specific folate metabolites and combinations thereof. PMID:19287311

  4. Genetic defects in folate and cobalamin pathways affecting the brain.

    PubMed

    Kirsch, Susanne H; Herrmann, Wolfgang; Obeid, Rima

    2013-01-01

    Folate and cobalamin are necessary for early brain development and function. Deficiency of folate or cobalamin during pregnancy can cause severe malformation in the central nervous system such as neural tube defects. After birth, folate and cobalamin deficiency can cause anemia, failure to thrive, recurrent infections, psychiatric and neurological symptoms. The folate and the homocysteine metabolic pathways interact at a central step where 5-methyltetrahydrofolate donates its methyl group to homocysteine to produce methionine and tetrahydrofolate. Methyl cobalamin and folate interact at this critical step. Both nutrients have a crucial role in DNA synthesis and in delivering S-adenosylmethionine, the universal methyl donor. Severe and mild inherited disorders in folate and cobalamin pathways have been described. The two groups of disorders share some similarities, but differ in the molecular mechanism, metabolic dysregulation, and disease management. This review summarizes selected disorders, including rare and common mutations that affect folate and cobalamin absorption, transport, or dependent enzymes. When the mutations are discovered early enough, many of the described disorders are easily treatable by B vitamin supplementation, which often prevents or reverses the manifestation of the disease. Therefore, the screening for mutations is recommended and should be carried out as early as possible: after occurrence of the first symptoms or when a certain constellations of the folate and cobalamin related markers are measured, such as elevated homocysteine and/or methylmalonic acid. PMID:23183749

  5. Mitochondrial MTHFD2L is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase expressed in both adult and embryonic tissues.

    PubMed

    Shin, Minhye; Bryant, Joshua D; Momb, Jessica; Appling, Dean R

    2014-05-30

    Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. We show here that the MTHFD2L enzyme is bifunctional, possessing both CH2-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. The dehydrogenase activity can use either NAD(+) or NADP(+) but requires both phosphate and Mg(2+) when using NAD(+). The NADP(+)-dependent dehydrogenase activity is inhibited by inorganic phosphate. MTHFD2L uses the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth. In adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung. PMID:24733394

  6. Folate and long-chain polyunsaturated fatty acids in psychiatric disease.

    PubMed

    Muskiet, Frits A J; Kemperman, Ramses F J

    2006-11-01

    Schizophrenia, autism and depression do not inherit by Mendel's law, and the search for a genetic basis seems unsuccessful. Schizophrenia and autism relate to low birth weight and pregnancy complications, which are associated with developmental adaptations by "programming". Epigenetics might constitute the basis of programming and depend on folate status and one-carbon metabolism in general. Early folate status of patients with schizophrenia might be compromised as suggested by (i) coinciding incidences of schizophrenia and neural tube defects (NTDs) in the Dutch hunger winter, (ii) coinciding seasonal fluctuations in birth of patients with schizophrenia and NTDs, (iii) higher schizophrenia incidence in immigrants and (iv) higher incidence in methylene tetrahydrofolate reductase 677C-->T homozygotes. Recent studies in schizophrenia and autism point at epigenetic silencing of critical genes or chromosomal loci. The long-chain polyunsaturated fatty acids (LCPUFA), arachidonic acid (AA, from meat) and docosahexaenoic acid (fish) are components of brain phospholipids and modulators of signal transduction and gene expression. Patients with schizophrenia and, possibly, autism exhibit abnormal phospholipid metabolism that might cause local AA depletion and impaired eicosanoid-mediated signal transduction. National fish intakes relate inversely with major and postpartum depressions. Five out of six randomized controlled trials with eicosapentaenoic acid (fish) have shown positive effects in schizophrenia, and 4 of 6 were favorable in depression and bipolar disorders. We conclude that folate and LCPUFA might be important in both the etiology and severity of at least some psychiatric diseases. PMID:16650750

  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. Binding cofactors with triplex-based DNA motifs.

    PubMed

    Kröner, Christoph; Göckel, Anja; Liu, Wenjing; Richert, Clemens

    2013-11-18

    Cofactors are pivotal compounds for the cell and many biotechnological processes. It is therefore interesting to ask how well cofactors can be bound by oligonucleotides designed not to convert but to store and release these biomolecules. Here we show that triplex-based DNA binding motifs can be used to bind nucleotides and cofactors, including NADH, FAD, SAM, acetyl CoA, and tetrahydrofolate (THF). Dissociation constants between 0.1 μM for SAM and 35 μM for THF were measured. A two-nucleotide gap still binds NADH. The selectivity for one ligand over the others can be changed by changing the sequence of the binding pocket. For example, a mismatch placed in one of the two triplets adjacent to the base-pairing site changes the selectivity, favoring the binding of FAD over that of ATP. Further, changing one of the two thymines of an A-binding motif to cytosine gives significant affinity for G, whereas changing the other does not. Immobilization of DNA motifs gives beads that store NADH. Exploratory experiments show that the beads release the cofactor upon warming to body temperature.

  9. 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.

  10. Type III mixed cryoglobulinemia and antiphospholipid syndrome in a patient with partial DiGeorge syndrome.

    PubMed

    Chang, Alice D; Tachdjian, Raffi; Gallagher, Kerry; McCurdy, Deborah K; Lassman, Charles; Stiehm, E Richard; Yadin, Ora

    2006-01-01

    We studied a 14 year-old boy with partial DiGeorge syndrome (DGS), status post complete repair of Tetralogy of Fallot, who developed antiphospholipid syndrome (APS) and type III mixed cryoglobulinemia. He presented with recurrent fever and dyspnea upon exertion secondary to right pulmonary embolus on chest computed tomography (CT). Coagulation studies revealed homozygous methylene tetrahydrofolate reductase 677TT mutations, elevated cardiolipin IgM antibodies, and elevated beta(2)-glycoprotein I IgM antibodies. Infectious work-up revealed only positive anti-streptolysin O (ASO) and anti-DNAse B titers. Autoimmune studies showed strongly positive anti-platelet IgM, elevated rheumatoid factor (RF), and positive cryocrit. Renal biopsy for evaluation of proteinuria and hematuria showed diffuse proliferative glomerulonephritis (DPGN) with membranoproliferative features consistent with cryoglobulinemia. Immunofixation showed polyclonal bands. Our patient was treated successfully with antibiotics, prednisone, and mycophenolate mofetil (MMF). This is the first report of a patient with partial DGS presenting with APS and type III mixed cryoglobulinemia possibly due to Streptococcal infection.

  11. Type III Mixed Cryoglobulinemia and Antiphospholipid Syndrome in a Patient With Partial DiGeorge Syndrome

    PubMed Central

    Chang, Alice D.; Tachdjian, Raffi; Gallagher, Kerry; McCurdy, Deborah K.; Lassman, Charles; Stiehm, E. Richard; Yadin, Ora

    2006-01-01

    We studied a 14 year-old boy with partial DiGeorge syndrome (DGS), status post complete repair of Tetralogy of Fallot, who developed antiphospholipid syndrome (APS) and type III mixed cryoglobulinemia. He presented with recurrent fever and dyspnea upon exertion secondary to right pulmonary embolus on chest computed tomography (CT). Coagulation studies revealed homozygous methylene tetrahydrofolate reductase 677TT mutations, elevated cardiolipin IgM antibodies, and elevated β2-glycoprotein I IgM antibodies. Infectious work-up revealed only positive anti-streptolysin O (ASO) and anti-DNAse B titers. Autoimmune studies showed strongly positive anti-platelet IgM, elevated rheumatoid factor (RF), and positive cryocrit. Renal biopsy for evaluation of proteinuria and hematuria showed diffuse proliferative glomerulonephritis (DPGN) with membranoproliferative features consistent with cryoglobulinemia. Immunofixation showed polyclonal bands. Our patient was treated successfully with antibiotics, prednisone, and mycophenolate mofetil (MMF). This is the first report of a patient with partial DGS presenting with APS and type III mixed cryoglobulinemia possibly due to Streptococcal infection. PMID:17162366

  12. 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

  13. [Justifying genetic and immune markers of efficiency and sensitivity under combined exposure to risk factors in mining industry workers].

    PubMed

    Dolgikh, O V; Zaitseva, N V; Krivtsov, A V; Gorshkova, K G; Lanin, D V; Bubnova, O A; Dianova, D G; Lykhina, T S; Vdovina, N A

    2014-01-01

    The authors evaluated and justified immunologic and genetic markers under combined exposure to risk factors in mining industry workers. Analysis covered polymorphism features of 29 genes with variant alleles possibly participating in occupationally conditioned diseases formation and serving as sensitivity markers of these diseases risk. The genes association selected demonstrates reliably changed polymorphism vs. the reference group (SOD2 superoxidedismutase gene, ANKK1 dophamine receptor gene, SULT1A1 sulphtransaminase gene, MTHFR methylene tetrahydrofolate reductase gene, VEGF endothelial growth factor gene, TNF-alpha tumor necrosis factor gene). Under combined exposure to occupational hazards (sylvinite dust, noise) in mining industry, this association can serve as adequate marking complex of sensitivity to development of occupationally conditioned diseases. Increased-production of immune cytokine regulation markers: tumor necrosis factor and vascular endothelial growth factor. Genes SOD2, ANKK1, SULT1A1, VEGF, TNFalpha are recommended as sensitivity markers, and the coded cytokines (tumor necrosis factor and endothelial growth factor) are proposed as effect markers in evaluation of health risk for workers in mining industry.

  14. Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate.

    PubMed

    Marks, Virginia D; van der Merwe, George K; van Vuuren, Hennie J J

    2003-05-01

    The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP. PMID:12689635

  15. 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

  16. 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.

  17. Immunoassay of 5-methyltetrahydrofolate: use of /sup 125/I-labeled protein A as the tracer molecule for specific antibody

    SciTech Connect

    Langone, J.J.

    1980-05-15

    A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. /sup 125/I-Labeled staphylococcal Protein A (/sup 125/I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of /sup 125/I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific; 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15 to 20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits.

  18. 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.

  19. Mammalian folylpoly-. gamma. -glutamate synthetase. 4. In vitro and in vivo metabolism of folates and analogues and regulation of folate homeostasis

    SciTech Connect

    Cook, J.D.; Cichowicz, D.J.; George, S.; Lawler, Ann; Shane, B.

    1987-01-27

    The regulation of folate and folate analogue metabolism was studied in vitro by using purified hog liver folylpolyglutamate synthetase as a model system and in vivo in cultured mammalian cells. The types of folylpolyglutamates that accumulate in vivo in hog liver, and changes in cellular folate levels and folylpolyglutamate distributions caused by physiological and nutritional factors such as changes in growth rates and methionine, folate, and vitamin B/sub 12/ status, can be mimicked in vitro by using purified enzyme. (/sup 3/H)Folylpolyglutamate distributions can be explained solely in terms of the substrate specificity of folylpolyglutamate synthetase and can be modeled by using kinetic parameters obtained with purified enzyme. Low levels of folylpolyglutamate synthetase activity are normally required for the cellular metabolism of folates to retainable polyglutamate forms, and consequently folate retention and concentration, while higher levels of activity are required for the synthesis of the long chain length derivatives that are found in mammalian tissues. The synthesis of very long chain derivatives, which requires tetrahydrofolate polyglutamates as substrates, is a very slow process in vivo. The slow metabolism of 5-methyltetrahydrofolate to retainable polyglutamate forms causes the decreased tissue retention of folate in B/sub 12/ deficiency. Although cellular folylpolyglutamate distributions change in response to nutritional and physiological modulations, it is unlikely that these changes play a regulatory role in one-carbon metabolism as folate distributions respond only slowly.

  20. Folate levels and polyglutamylation profiles of papaya (Carica papaya cv. Maradol) during fruit development and ripening.

    PubMed

    Ramos-Parra, Perla A; García-Salinas, Carolina; Hernández-Brenes, Carmen; de la Garza, Rocío I Díaz

    2013-04-24

    Folates are essential micronutrients for humans, and their deficiency causes several detrimental effects on human health. Papaya fruit is an important natural source of some micronutrients. This paper presents a first complete characterization of folate derivatives accumulated in cv. Maradol papaya during fruit development and ripening processes. During postharvest ripening, the fruit accumulated up to 24.5% of the daily folate recommended dietary allowance (RDA) for an adult in a 1 cup (145 g) portion. Tetrahydrofolate (THF) and 5-methyl-THF were the predominant folate classes observed. Surprisingly, an unusually long polyglutamylation profile of tentatively up to 17 glutamates linked to 5-methyl-THF was detected; to the authors' knowledge, this very long polyglutamyl tail has not been reported for any organism, and it is probably characteristic of this plant species. This polyglutamylation degree changed throughout fruit development and ripening, showing the largest differences at the onset of ripening. This work raises questions about the functional role of folate derivatives in fruit development.

  1. Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis.

    PubMed

    Field, Martha S; Kamynina, Elena; Watkins, David; Rosenblatt, David S; Stover, Patrick J

    2015-01-13

    An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

  2. 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.

  3. Sign epistasis limits evolutionary trade-offs at the confluence of single- and multi-carbon metabolism in Methylobacterium extorquens AM1.

    PubMed

    Carroll, Sean Michael; Lee, Ming-Chun; Marx, Christopher J

    2014-03-01

    Adaptation of one set of traits is often accompanied by attenuation of traits important in other selective environments, leading to fitness trade-offs. The mechanisms that either promote or prevent the emergence of trade-offs remain largely unknown, and are difficult to discern in most systems. Here, we investigate the basis of trade-offs that emerged during experimental evolution of Methylobacterium extorquens AM1 to distinct growth substrates. After 1500 generations of adaptation to a multi-carbon substrate, succinate (S), many lineages had lost the ability to use one-carbon compounds such as methanol (M), generating a mixture of M(+) and M(-) evolved phenotypes. We show that trade-offs in M(-) strains consistently arise via antagonistic pleiotropy through recurrent selection for loss-of-function mutations to ftfL (formate-tetrahydrofolate ligase), which improved growth on S while simultaneously eliminating growth on M. But if loss of FtfL was beneficial, why were M trade-offs not found in all populations? We discovered that eliminating FtfL was not universally beneficial on S, as it was neutral or even deleterious in certain evolved lineages that remained M(+) . This suggests that sign epistasis with earlier arising mutations prevented the emergence of mutations that drove trade-offs through antagonistic pleiotropy, limiting the evolution of metabolic specialists in some populations.

  4. Disentangling fetal and maternal susceptibility for pre-eclampsia: a British multicenter candidate-gene study.

    PubMed

    2005-07-01

    The Genetics of Pre-Eclampsia (GOPEC) collaboration aims to identify genetic factors in U.K. families affected by pre-eclampsia. A number of genetic studies have reported associations with pre-eclampsia, but attempts to replicate these findings have yielded inconsistent results. We describe the results of extensive genotyping of seven candidate genes previously reported as conferring susceptibility to pre-eclampsia. Six hundred fifty-seven women affected by pre-eclampsia and their families were genotyped at 28 single-nucleotide polymorphisms in the genes encoding angiotensinogen, the angiotensin receptors, factor V Leiden variant, methylene tetrahydrofolate reductase, nitric oxide synthase, and TNFalpha. Genotypes were analyzed by the transmission/disequilibrium test. Genotype risk ratios (GRRs) associated with maternal genotypes had a range of 0.70-1.16; GRRs associated with fetal genotypes had a range of 0.72-1.11. No GRR achieved the prespecified criteria for statistical significance (posterior probability >.05). We conclude that none of the genetic variants tested in this large study of strictly defined pre-eclamptic pregnancies confers a high risk of disease. The results emphasize the importance of conducting rigorously designed studies of adequate size to provide precise genetic risks with narrow confidence intervals, if overreporting of false-positive results is to be avoided.

  5. Nuclear receptor-mediated alleviation of alcoholic fatty liver by polyphenols contained in alcoholic beverages.

    PubMed

    Yao, Ruiqing; Yasuoka, Akihito; Kamei, Asuka; Ushiama, Shota; Kitagawa, Yoshinori; Rogi, Tomohiro; Shibata, Hiroshi; Abe, Keiko; Misaka, Takumi

    2014-01-01

    To elucidate the effect of the polyphenols contained in alcoholic beverages on the metabolic stress induced by ethanol consumption, four groups of mice were fed for five weeks on Lieber's diet with or without ethanol, with ethanol plus ellagic acid, and with ethanol plus trans-resveratrol. Alcoholic fatty liver was observed in the group fed the ethanol diet but not in those fed the ethanol plus polyphenol diets. Liver transcriptome analysis revealed that the addition of the polyphenols suppressed the expression of the genes related to cell stress that were up-regulated by ethanol alone. Conversely, the polyphenols up-regulated the genes involved in bile acid synthesis, unsaturated fatty acid elongation, and tetrahydrofolate synthesis that were down-regulated by ethanol alone. Because parts of these genes were known to be regulated by the constitutive androstane receptor (CAR), we performed the same experiment in the CAR-deficient mice. As a result, fatty liver was observed not only in the ethanol group but also with the ethanol plus polyphenol groups. In addition, there was no segregation of the gene expression profiles among these groups. These results provide a molecular basis for the prevention of alcohol-induced stress by the polyphenols in alcoholic beverages.

  6. 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

  7. 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.

  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. ABO blood group but not haemostasis genetic polymorphisms significantly influence thrombotic risk: a study of 180 homozygotes for the Factor V Leiden mutation.

    PubMed

    2006-12-01

    Limited data exist on the impact of additional genetic risk factors on the clinical manifestations of factor (F) V Leiden homozygotes. A retrospective multi-centre cohort study was performed to assess the role of the FII G20210A gene mutation, the protein C (PC) promoter CG haplotype, the combination of two PC polymorphisms (A-1641G, C-1654T), the FXIII Val34Leu polymorphism, two thrombin-activatable fibrinolysis inhibitor polymorphisms (Thr325Ile, Ala147Thr), two plasminogen activator inhibitor-1 polymorphisms (-675 4G/5G, A-844G), the methylene-tetrahydrofolate reductase (MTHFR) C677T polymorphism and the ABO blood group on the thrombotic phenotype in FV Leiden homozygotes. 127 subjects with venous thrombosis and 53 asymptomatic subjects were analysed. The T allele of MTHFR C677T was more frequent in symptomatic subjects than in asymptomatic ones (68% vs. 45%, P = 0.02; odds ratio (OR) 2.8, 95% CI 1.3-5.8, after adjustment for potential confounders). For the other polymorphisms, no difference was observed between symptomatic and asymptomatic subjects. The non-O blood group was more frequent among symptomatic carriers (84% vs. 57%, P = 0.0002; OR 4.1, 95% CI 1.7-9.7). In conclusion, except for the ABO blood group, none of the polymorphisms studied contribute strongly to the thrombotic risk in FV Leiden homozygotes.

  10. 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

  11. 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.

  12. 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

  13. Metabolism of one-carbon compounds by the ruminal acetogan syntrophococcus sucromutans

    SciTech Connect

    Dore, J.; Bryant, M.P. )

    1990-04-01

    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 (Woods) 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-{sup 14}C) pyruvate, and chemically synthesized O-(methyl-{sup 14}C) vanillate were used to further investigate the catabolism of one-carbon (C{sub 1}) 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 CO{sub 2} was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO{sub 2} readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO{sub 2} and pyruvate predominantly labeled the carboxyl group acetate. No CO{sub 2} 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.

  14. Evidence by ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones

    PubMed Central

    Gaikwad, Nilesh W.; Rogan, Eleanor G.; Cavalieri, Ercole L.

    2007-01-01

    Estrogen ortho-quinones have been implicated as ultimate carcinogenic metabolites of estrogens. The present conclusion that estrogen ortho-quinones are not substrates for NAD(P)H:quinone oxidoreductase (NQO1) stems from earlier reports. In this investigation, we were successful in circumventing the problem of nonenzymatic reduction of estrogen quinone by NAD(P)H, which led to the above conclusion, and for the first time show that NQO1 catalyzes the reduction of estrogen quinones. Mass spectrometric binding studies involving estradiol-3,4-quinone or menadione with NQO1 clearly support the formation of an enzyme-substrate physical complex. However, the NQO1 mass spectrum did not alter after addition of cholesterol, the control. Two different strategies were employed to ascertain the NQO1 activity in estrogen quinone reduction. First, the ping-pong mechanism of NQO1 catalysis was utilized to overcome the problem of nonenzymatic reduction of the substrate by NADH. Second, tetrahydrofolic acid, which has a lower reducing potential, was used as an alternate cofactor. Both of these methods confirmed the reduction of estradiol-3,4-quinone by NQO1, when assay mixtures were analyzed by UV or liquid chromatography-mass spectrometry. Furthermore, reduction of 9,10-phenanthrene quinone or menadione was observed using the reported assay conditions. Thus, clear evidence for the catalytic reduction of estrogen ortho-quinones by NQO1 has been obtained; its mechanism and implications are discussed. PMID:17893042

  15. Mitochondrial function and toxicity: role of B vitamins on the one-carbon transfer pathways.

    PubMed

    Depeint, Flore; Bruce, W Robert; Shangari, Nandita; Mehta, Rhea; O'Brien, Peter J

    2006-10-27

    The B vitamins are water-soluble vitamins that are required as coenzymes for reactions essential for cellular function. This review focuses on the essential role of vitamins in maintaining the one-carbon transfer cycles. Folate and choline are believed to be central methyl donors required for mitochondrial protein and nucleic acid synthesis through their active forms, 5-methyltetrahydrofolate and betaine, respectively. Cobalamin (B12) may assist methyltetrahydrofolate in the synthesis of methionine, a cysteine source for glutathione biosynthesis. Pyridoxal, pyridoxine and pyridoxamine (B6) seem to be involved in the regeneration of tetrahydrofolate into the active methyl-bearing form and in glutathione biosynthesis from homocysteine. Other roles of these vitamins that are relevant to mitochondrial functions will also be discussed. However these roles for B vitamins in cell function are mostly theoretically based and still require verification at the cellular level. For instance it is still not known what B vitamins are depleted by xenobiotic toxins or which cellular targets, metabolic pathways or molecular toxic mechanisms are prevented by B vitamins. This review covers the current state of knowledge and suggests where this research field is heading so as to better understand the role vitamin Bs play in cellular function and intermediary metabolism as well as molecular, cellular and clinical consequences of vitamin deficiency. The current experimental and clinical evidence that supplementation alleviates deficiency symptoms as well as the effectiveness of vitamins as antioxidants will also be reviewed.

  16. Formyltetrahydrofolate synthetase gene diversity in the guts of higher termites with different diets and lifestyles.

    PubMed

    Ottesen, Elizabeth A; Leadbetter, Jared R

    2011-05-01

    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.

  17. Nuclear Receptor-Mediated Alleviation of Alcoholic Fatty Liver by Polyphenols Contained in Alcoholic Beverages

    PubMed Central

    Yao, Ruiqing; Yasuoka, Akihito; Kamei, Asuka; Ushiama, Shota; Kitagawa, Yoshinori; Rogi, Tomohiro; Shibata, Hiroshi; Abe, Keiko; Misaka, Takumi

    2014-01-01

    To elucidate the effect of the polyphenols contained in alcoholic beverages on the metabolic stress induced by ethanol consumption, four groups of mice were fed for five weeks on Lieber's diet with or without ethanol, with ethanol plus ellagic acid, and with ethanol plus trans-resveratrol. Alcoholic fatty liver was observed in the group fed the ethanol diet but not in those fed the ethanol plus polyphenol diets. Liver transcriptome analysis revealed that the addition of the polyphenols suppressed the expression of the genes related to cell stress that were up-regulated by ethanol alone. Conversely, the polyphenols up-regulated the genes involved in bile acid synthesis, unsaturated fatty acid elongation, and tetrahydrofolate synthesis that were down-regulated by ethanol alone. Because parts of these genes were known to be regulated by the constitutive androstane receptor (CAR), we performed the same experiment in the CAR-deficient mice. As a result, fatty liver was observed not only in the ethanol group but also with the ethanol plus polyphenol groups. In addition, there was no segregation of the gene expression profiles among these groups. These results provide a molecular basis for the prevention of alcohol-induced stress by the polyphenols in alcoholic beverages. PMID:24498295

  18. Genome Characteristics of Two Novel Type I Methanotrophs Enriched from North Sea Sediments Containing Exclusively a Lanthanide-Dependent XoxF5-Type Methanol Dehydrogenase.

    PubMed

    Vekeman, Bram; Speth, Daan; Wille, Jasper; Cremers, Geert; De Vos, Paul; Op den Camp, Huub J M; Heylen, Kim

    2016-10-01

    Microbial methane oxidizers play a crucial role in the oxidation of methane in marine ecosystems, as such preventing the escape of excessive methane to the atmosphere. Despite the important role of methanotrophs in marine ecosystems, only a limited number of isolates are described, with only four genomes available. Here, we report on two genomes of gammaproteobacterial methanotroph cultures, affiliated with the deep-sea cluster 2, obtained from North Sea sediment. Initial enrichments using methane as sole source of carbon and energy and mimicking the in situ conditions followed by serial subcultivations and multiple extinction culturing events over a period of 3 years resulted in a highly enriched culture. The draft genomes of the methane oxidizer in both cultures showed the presence of genes typically found in type I methanotrophs, including genes encoding particulate methane monooxygenase (pmoCAB), genes for tetrahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, and genes of the ribulose monophosphate (RuMP) pathway. The most distinctive feature, when compared to other available gammaproteobacterial genomes, is the absence of a calcium-dependent methanol dehydrogenase. Both genomes reported here only have a xoxF gene encoding a lanthanide-dependent XoxF5-type methanol dehydrogenase. Thus, these genomes offer novel insight in the genomic landscape of uncultured diversity of marine methanotrophs. PMID:27457652

  19. Increased plasma homocysteine levels in shift working bus drivers

    PubMed Central

    Martins, P; D'Almeida, V; Vergani, N; Perez, A; Tufik, S

    2003-01-01

    Background: Previous studies have indicated an association between shift work and cardiovascular disease. There is also considerable epidemiological evidence that hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Aims: To analyse plasma homocysteine levels in shift work bus drivers, and to investigate possible relations with sleep parameters and other biochemical factors. Methods: Blood samples were collected from 30 male shift working long-haul bus drivers in a Brazilian sample and analysed for plasma levels of homocysteine, folic acid, vitamin B12, and serum lipids. A group of 22 daytime workers, matched for age and body mass index served as controls. The incidence of mutations in the gene coding for methylene tetrahydrofolate, an enzyme which is related to hyperhomocysteinemia, was also assessed. Polysomnographic recordings were obtained from the target group. Results: Bus drivers showed significantly higher levels of plasma homocysteine than the control group (18.57 v 9.43 µM). Most of the other biochemical, behavioural, and molecular parameters did not differ between groups. Likewise, sleep parameters appeared to be within the normal range. Conclusions: The significantly increased plasma homocysteine levels in long-haul bus drivers did not appear to be secondary to other biochemical or behavioural problems in this group. These results suggest that hyperhomocysteinemia may be involved in the increased incidence of cardiovascular diseases observed in shift workers. PMID:12937187

  20. Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs.

    PubMed Central

    Eastman, H B; Swick, A G; Schmitt, M C; Azizkhan, J C

    1991-01-01

    Dihydrofolate reductase (DHFR; EC 1.5.1.3) is required in folate metabolism for the synthesis of purines, thymidine, and glycine. Although there have been several reports of induction of DHFR enzyme by methotrexate (MTX), a drug that competitively inhibits DHFR, there are no studies reported that examine the effect of MTX on DHFR gene transcription. We have examined the effect of MTX and other inhibitors of DNA synthesis on DHFR transcription using a transient expression assay. MTX stimulates transient expression in a concentration-dependent manner from a hamster DHFR promoter construct containing 150 base pairs 5' to the start of transcription. Addition of either tetrahydrofolate or hypoxanthine plus thymidine prevents the promoter induction in response to MTX, suggesting that stimulation by MTX results from inhibition of these metabolites. Furthermore, two other antimetabolic drugs--fluorodeoxyuridine and hydroxyurea--also stimulate the DHFR promoter in a concentration-dependent manner. In contrast, aphidicolin, which blocks cell growth through inhibition of DNA polymerase alpha, has no effect on the DHFR promoter. The potential relevance of these results to cross-resistance to chemotherapeutic agents and to the process of gene amplification is discussed. Images PMID:1833762

  1. 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

  2. Veratrol-O-demethylase of Acetobacterium dehalogenans: ATP-dependent reduction of the corrinoid protein.

    PubMed

    Siebert, Anke; Schubert, Torsten; Engelmann, Tina; Studenik, Sandra; Diekert, Gabriele

    2005-09-01

    The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co(I)] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E (SHE)) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be -290 mV (pH 7.5), whereas E (SHE) at pH 7.5 was lower than -450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5'-(beta,gamma-imido)triphosphate (AMP-PNP, 2-4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM). PMID:15968525

  3. [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

  4. 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

  5. Prevalence of MTHFR C677T polymorphism in north Indian mothers having babies with Trisomy 21 Down syndrome.

    PubMed

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

    2008-10-01

    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 examined the prevalence of the MTHFR C677T polymorphism among 104 north Indian mothers of babies with Down syndrome and 109 control mothers. The prevalence of MTHFR C677T polymorphism observed among mothers of babies with Down syndrome was 28% compared to 35% in controls (C677T/T677T). There was no significant difference between the two groups (p = 0.294). Mean homocysteine level in mothers of children with Down syndrome was lower than the level in the controls. Our data suggests that the MTHFR C677T polymorphism is not associated with an increased risk of Down syndrome in the north Indian population. Homocysteine levels in our study were higher when compared to other studies. Methylcobolamin and folate deficiency or use of random samples for homocysteine determination could possibly account for this observation.

  6. 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

  7. 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

  8. 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

  9. Folate levels and polyglutamylation profiles of papaya (Carica papaya cv. Maradol) during fruit development and ripening.

    PubMed

    Ramos-Parra, Perla A; García-Salinas, Carolina; Hernández-Brenes, Carmen; de la Garza, Rocío I Díaz

    2013-04-24

    Folates are essential micronutrients for humans, and their deficiency causes several detrimental effects on human health. Papaya fruit is an important natural source of some micronutrients. This paper presents a first complete characterization of folate derivatives accumulated in cv. Maradol papaya during fruit development and ripening processes. During postharvest ripening, the fruit accumulated up to 24.5% of the daily folate recommended dietary allowance (RDA) for an adult in a 1 cup (145 g) portion. Tetrahydrofolate (THF) and 5-methyl-THF were the predominant folate classes observed. Surprisingly, an unusually long polyglutamylation profile of tentatively up to 17 glutamates linked to 5-methyl-THF was detected; to the authors' knowledge, this very long polyglutamyl tail has not been reported for any organism, and it is probably characteristic of this plant species. This polyglutamylation degree changed throughout fruit development and ripening, showing the largest differences at the onset of ripening. This work raises questions about the functional role of folate derivatives in fruit development. PMID:23574547

  10. Acute bilateral vision loss in emergency department: A case report.

    PubMed

    Tanrikulu, Ceren Sen; Hocagil, Hilal; Kaya, Ural; Hocagil, Abdullah Cuneyt

    2016-03-01

    Stroke occurs due to the interruption of blood flow to the brain and it is divided into ischemic and hemorrhagic. In the ischemic strokes, while the most commonly affected vessel is median cerebral artery (MCA), it is particularly affected bilateral posterior cerebral artery (PCA) is very rare condition. In this study, a case of sudden loss of vision and bilateral occipital infarct associated with bilateral vertebral system pathology and methylene tetrahydrofolate reductase (MTHFR) gene mutation were reported. A 62-year-old man was admitted with sudden loss of vision complaint starting 10 h before applying to emergency department. The patient was oriented and cooperative. On neurological examination, there was complete loss of vision in the right eye and only a response to light in the left eye. On the brain computerized tomography (CT), ischemic lesions were observed in the bilateral occipital areas and on magnetic resonance imaging (MRI), there were foci showing diffusion limitation in cortico-subcortical areas of bilateral parieto-occipital region. On the detailed examination at the clinic, MTHFR (a1298c) gene mutation was detected. Bilateral occipital infarction is rare and its diagnosis can be difficult because of its atypical symptoms. Therefore, occipital infarction should be suspected when the only sign is isolated vision loss in patients with risk factor for thromboembolism in their history and detailed visual-neurological examination of these patients should be performed. PMID:27239639

  11. Expression of genes encoding enzymes involved in the one carbon cycle in rat placenta is determined by maternal micronutrients (folic acid, vitamin B12) and omega-3 fatty acids.

    PubMed

    Khot, Vinita; Kale, Anvita; Joshi, Asmita; Chavan-Gautam, Preeti; Joshi, Sadhana

    2014-01-01

    We have reported that folic acid, vitamin B12, and omega-3 fatty acids are interlinked in the one carbon cycle and have implications for fetal programming. Our earlier studies demonstrate that an imbalance in maternal micronutrients influence long chain polyunsaturated fatty acid metabolism and global methylation in rat placenta. We hypothesize that these changes are mediated through micronutrient dependent regulation of enzymes in one carbon cycle. Pregnant dams were assigned to six dietary groups with varying folic acid and vitamin B12 levels. Vitamin B12 deficient groups were supplemented with omega-3 fatty acid. Placental mRNA levels of enzymes, levels of phospholipids, and glutathione were determined. Results suggest that maternal micronutrient imbalance (excess folic acid with vitamin B12 deficiency) leads to lower mRNA levels of methylene tetrahydrofolate reductase (MTHFR) and methionine synthase , but higher cystathionine b-synthase (CBS) and Phosphatidylethanolamine-N-methyltransferase (PEMT) as compared to control. Omega-3 supplementation normalized CBS and MTHFR mRNA levels. Increased placental phosphatidylethanolamine (PE), phosphatidylcholine (PC), in the same group was also observed. Our data suggests that adverse effects of a maternal micronutrient imbalanced diet may be due to differential regulation of key genes encoding enzymes in one carbon cycle and omega-3 supplementation may ameliorate most of these changes.

  12. Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

    PubMed Central

    Di Salvo, Martino L.; Scarsdale, J. Neel; Kazanina, Galina; Contestabile, Roberto; Schirch, Verne; Wright, H. Tonie

    2013-01-01

    Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs. PMID:23956983

  13. LC-MS based assay to measure intracellular compound levels in Mycobacterium smegmatis: linking compound levels to cellular potency.

    PubMed

    Bhat, Jyothi; Narayan, Ashwini; Venkatraman, Janani; Chatterji, Monalisa

    2013-08-01

    Dihydrofolate reductase (DHFR) plays a central role in maintaining cellular pool of tetrahydrofolic acid, a cofactor necessary for DNA, RNA and protein synthesis. The clinical validation of DHFR as antibacterial target was established by the success of trimethoprim (TMP). DHFR is also an attractive target for identifying anti-tuberculosis molecules however, due to observed weak cellular potency, no DHFR inhibitors have been developed as drugs so far. TMP and its analogs have poor cellular potency on Mycobacterium tuberculosis and Mycobacterium smegmatis cells. We found a mutant strain of M. smegmatis, mc²155 to be sensitive to TMP whereas wild type strain was not inhibited by TMP. We utilized this system to probe if poor or lack of activity of TMP is a consequence of poor intracellular compound levels. An LC-MS based method was developed for measuring TMP and rifampicin (RIF) in M. smegmatis. Using the assay, equivalent RIF levels were observed in both strains however, TMP was detected only in mc²155 cells, hence proving a positive correlation between potency and compound levels. To the best of our knowledge this is the first time LC-MS method has been used to measure compound levels in mycobacterial cells. We propose it to be a valuable tool to understand the lack of potency or resistance mechanisms in antimycobacterial drug development.

  14. 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.

  15. Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

    PubMed Central

    Ariel, M; Lavi, H; Holtzer, E; Grossowicz, N

    1982-01-01

    We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent. PMID:6978334

  16. Concentration-Dependent Processivity of Multiple Glutamate Ligations Catalyzed by Folylpoly-γ-glutamate Synthetase

    PubMed Central

    Tomsho, John W.; Moran, Richard G.; Coward, James K.

    2010-01-01

    Folylpoly-γ-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-γ-glutamate derivatives of reduced folates and anti-folates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH4PteGlu1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH4PteGlu1 catalyzed by FPGS in order to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to measure folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues onto DDAH4PteGlu1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells. PMID:18672898

  17. A critical intracellular concentration of fully reduced non-methylated folate polyglutamates prevents macrocytosis and diminished growth rate of human cell line K562 in culture.

    PubMed Central

    Watkins, D; Cooper, B A

    1983-01-01

    Growth rate of human leukaemic cell line K562 was independent of intracellular folate concentration when this was greater than 1.5 microM. When intracellular folate concentration was less than 1.5 microM, the rate of growth was proportional to the logarithm of intracellular concentration of non-methylated fully reduced folates, but not to the logarithm of the intracellular concentration of N5-methyltetrahydropteroylglutamate. Intracellular folate concentration sufficient to support an optimal growth rate was maintained by either DL-N5-formyltetrahydropteroylglutamate or DL-N5-methyltetrahydropteroylglutamate at a 100-fold lower concentration than pteroylglutamate. Addition of hypoxanthine to culture medium partially restored growth of folate-depleted cells: thymidine had no effect on growth rate either alone or in combination with thymidine. Folate-depleted cells with diminished growth rate were larger than replete cells, but did not have megaloblastic morphology. The mitotic index was not decreased in cultures with diminished growth rate. The rate of growth and cell size of K562 cells is thus dependent on a critical intracellular concentration of non-methylated tetrahydrofolates, which may be maintained by different concentrations of either reduced folates or pteroylglutamate. PMID:6577860

  18. 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.

  19. Four Components of the Conjugated Redox System in Organisms: Carbon, Nitrogen, Sulfur, Oxygen.

    PubMed

    Tereshina, E V; Laskavy, V N; Ivanenko, S I

    2015-09-01

    C1 compounds participate in various metabolic processes and regulations including DNA methylation. Formaldehyde (FA), a product of methyl group oxidation, is highly cytotoxic. In the cell, there are two pathways of its utilization: assimilation and oxidation. Formaldehyde displays cytotoxicity, and therefore its oxidation is considered as detoxification. The sensitivity to the threshold concentration of FA we regard as an indication of its major role in biosystem functioning. A model of a three-component conjugated redox system is proposed in which the methyl group oxidation pathway is an archaic and conservative donor of protons and electrons, the reduction of O2 serves as an acceptor, and the arginine amino group is used for production of both urea and nitric oxide (the donor and acceptor, respectively). The fourth component of the redox system is glutathione, which maintains redox balance. The three-level system of proton donors includes the oxidation of a methyl group (first level), the oxidation of acetate in mitochondria (second level), and glucose catabolism in the pentose phosphate pathway (third level). The whole redox system is united by the sulfhydryl groups of cysteines, glutathione, thioredoxin, and α-lipoic acid. The central regulatory role in this redox system belongs to glutathione-dependent formaldehyde dehydrogenase, which controls FA binding with tetrahydrofolic acid, arginine methylation, and denitrosation of sulfhydryl groups. The conjugated redox system was formed during evolution as a union of separate redox cycles of carbon, nitrogen, sulfur, and oxygen.

  20. Design, synthesis, and enzyme kinetics of novel benzimidazole and quinoxaline derivatives as methionine synthase inhibitors.

    PubMed

    Elshihawy, Hosam; Helal, Mohamed A; Said, Mohamed; Hammad, Mohamed A

    2014-01-01

    Methionine synthase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate. Benzimidazole and deazatetrahydrofolates derivatives have been shown to inhibit methionine synthase by competing with the substrate 5-methyltetrahydrofolate. In this study, a novel series of substituted benzimidazoles and quinoxalines were designed and assessed for inhibitory activity against purified rat liver methionine synthase using a radiometric enzyme assay. Compounds 3g, 3j, and 5c showed the highest activity against methionine synthase (IC₅₀: 20 μM, 18 μM, 9 μM, respectively). Kinetic analysis of these compounds using Lineweaver-Burk plots revealed characteristics of mixed inhibition for 3g and 5c; and uncompetitive inhibition for 3j. Docking study into a homology model of the rat methionine synthase gave insights into the molecular determinants of the activity of this class of compounds. The identification of these drug-like inhibitors could lead the design of the next generation modulators of methionine synthase.

  1. [Recent advances in Sphingobium sp. SYK-6 for lignin aromatic compounds degradation--a review].

    PubMed

    Zhang, Xiaoyan; Peng, Xue; Masai, Eiji

    2014-08-01

    Lignin is complex heteropolymer produced from hydroxycinnamyl alcohols through radical coupling. In nature, white-rot fungi are assumed initially to attack native lignin and release lignin-derived-low-molecular-weight compounds, and soil bacteria play an importent role for completely degradation of these compounds. Study on the soil bacteria degrading lignin-derived-low-molecular-weight compounds will give way to understand how aromatic compounds recycle in nature, and to utilize lignin compounds as the renewable materials for valuable materials production. Sphingobium sp. SYK-6 that grows on lignin biphenyl (5,5'-dehydrodivanillate) had been isolated from pulp effluent in 1987. We have researched this bacterium more than 25 years, a serious aromatic metabolic pathway has been determined, and related genes have been isolated. As the complete genome sequence of SYK-6 has been opened to the public in 2012, the entire aromatic compounds degradation mechanisms become more clear. Main contents in our review cover: (1) genome information; (2) aryl metabolism; (3) biphenyl metabolism; (4) ferulate metabolism; (5) tetrahydrofolate-dependent O-demethylation system for lignin compound degrdation; (6) protocatechuate 4,5-cleavage pathway; (7) multiple pathways for 3-O-methylgallate metabolism.

  2. [Biochemical modulation of 5-fluorouracil by high-dose leucovorin].

    PubMed

    Aiba, K

    1988-03-01

    The biochemical modulation of 5-fluorouracil (FU) by using it in combination with leucovorin (LV) is reviewed. One of the mechanisms of action of FU is inhibition of thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate required for DNA repair and synthesis. In the presence of 5, 10-methylene tetrahydrofolate, 5-fluoro-deoxyuridine monophosphate (FdUMP), which is an active nucleotide of FU, forms a ternary complex with TS, resulting in enzyme inhibition. The rationale for the use of LV with FU is to increase of formation and stability of catalytically inactive ternary complexes. In clinical trials, the dose schedules of 500 mg/m2 iv bolus or 500 mg/m2 continuously iv infusion is frequently used in order to obtain an ideal plasma concentration of active LV metabolites. Preliminary clinical trials using the combination of FU and LV appear to be capable of increasing of the clinical activity of FU for colorectal and breast cancers. Several randomized studies have supported the increase in response rates seen in the uncontrolled trials but additional studies are required to demonstrate a positive impact on the survival of treated patients.

  3. Nature of Transient Inhibition of Deoxyribonucleic Acid Synthesis in HeLa Cells by Parainfluenza Virus 1 (Sendai)

    PubMed Central

    Fuchs, Pinhas; Kohn, Alexander

    1971-01-01

    Adsorption of ultraviolet-inactivated Sendai virus, at high or low multiplicity, to HeLa cells caused a transient increased incorporation of 3H-thymidine into the cellular deoxyribonucleic acid (DNA). In HeLa cells synchronized by a double-thymidine block, this increased incorporation of thymidine during the S phase lasted from about 30 to 90 min after virus adsorption. The observations that the kinetics of accumulation of radioactive thymidine in the nucleotide pool did not differ in control and in the virus-treated cells and that the 32P incorporation into the DNA of the virus-treated cells was inhibited at the same time indicate that the augmented incorporation of 3H-thymidine into DNA results from a transient block in the endogenous pathway of thymidine synthesis. Chromatographic analysis of the nucleotide pool of the virus-treated cells labeled with 14C-formate indicates that methylation of deoxyuridine monophosphate to thymidine monophosphate is inhibited. It is suggested that the inhibition is caused by a block of either the thymidilate synthetase or some step in the tetrahydrofolate cycle. PMID:4332139

  4. 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

  5. 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

  6. Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules.

    PubMed

    Boland, M J; Hanks, J F; Reynolds, P H; Blevins, D G; Tolbert, N E; Schubert, K R

    1982-06-01

    Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

  7. Thrombosis and occlusion of vascular access in hemodialyzed patients.

    PubMed

    Montagnana, Martina; Meschi, Tiziana; Borghi, Loris; Lippi, Giuseppe

    2011-11-01

    Patients undergoing chronic hemodialysis have a high risk of arterial thrombotic events as well as vascular access thrombosis (VAT). The latter complication has been consistently associated with inherited (i.e., the prothrombin 20210 polymorphism, and polymorphisms in the genes encoding for transforming growth factor-β1, nitric oxide synthase, plasminogen activator inhibitor-1, angiotensin converting enzyme, and methylene tetrahydrofolate reductase), and acquired thrombotic risk factors (i.e., diabetes, obesity, atrial fibrillation, hypertension, hyperhomocysteinemia, hyperlipoproteinemia(a), low serum albumin, antiphospholipid antibodies, autoantibodies against protein C and S, erythropoietin administration, malnutrition, and cytomegalovirus infection). The three main factors involved in the pathogenesis of VAT overlap those of venous thrombosis and therefore include endothelial cell injury, blood stasis, and hypercoagulability. These changes are characteristic of patients affected by end-stage renal disease and might be further aggravated during and after hemodialysis. The aim of this review is to describe the epidemiology and pathogenesis of thrombosis of dialysis vascular access and to discuss the application of therapeutic interventions in prevention and treatment of this clinical problem. PMID:22198859

  8. 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

  9. 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

  10. Manipulating the sulfur amino acid content of the early diet and its implications for long-term health.

    PubMed

    Rees, William D

    2002-02-01

    Epidemiological studies of human populations show that poor growth in utero predisposes an individual to the later development of type 2 (non-insulin-dependent) diabetes mellitus and hypertension in adulthood. This phenomenon is not confined to man; feeding pregnant rats diets moderately deficient in protein has a similar effect, programming the adult blood pressure and glucose metabolism of the offspring. A restriction in the amino acid supply was thought to cause poor fetal growth. However, recent experiments have shown that this is not the case and instead have implicated the metabolism of the S-containing amino acids. Many semi-synthetic experimental diets contain an imbalance in S-containing amino acids, forcing the animal to synthesise a sizeable part of its cysteine requirement from methionine. Unfortunately, when the diet is low in protein, the oxidation of amino acids is reduced, perturbing methionine metabolism and increasing levels of homocysteine. It is this interaction between protein content and composition of the diet which influences neonatal viability and may also determine the long-term health of the offspring. An excess of homocysteine is known to affect levels of two of the main mediators of cellular methylation reactions, S-adenosyl methionine and methylene tetrahydrofolate. S-adenosyl methionine is the methyl donor for the methylation of newly-synthesised DNA, regulating chromatin assembly and gene expression. The balance between S-adenosyl methionine and the methylated derivatives of folic acid may be critical for the development of differentiating cells and the long-term regulation of gene expression.

  11. [Purine regulon of gamma-proteobacteria: a detailed description].

    PubMed

    Ravcheev, D A; Gel'fand, M S; Mironov, A A; Rakhmaninova, A B

    2002-09-01

    The structure of the purine regulon was studied by a comparative genomic approach in seven genomes of gamma-proteobacteria: Escherichia coli, Salmonella typhi, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Vibrio cholerae. The palindromic binding site of the purine repressor (consensus ACGCAAACGTTTGCGT) is fairly well retained of genes encoding enzymes that participate in the synthesis of inosinemonophosphate from phosphoribozylpyrophosphate and in transfer of unicarbon groups, and also upstream of some transport protein genes. These genes may be regarded as the main part of the purine regulon. In terms of physiology, the regulation of the purC and gcvTHP/folD genes seems to be especially important, because the PurR site was found upstream of nonorthologous but functionally replaceable genes. However, the PurR site is poorly retained in front of orthologs of some genes belonging to the E. coli purine regulon, such as genes involved in general nitrogen metabolism, biosynthesis of pyrimidines, and synthesis of AMP and GMP from IMP, and also upstream of the purine repressor gene. It is predicted that purine regulons of the examined bacteria include the following genes: upp participating in synthesis of pyrimidines; uraA encoding an uracil transporter gene; serA involved in serine biosynthesis; folD responsible for the conversion of N5,N10-methenyl tetrahydrofolate into N10-formyltetrahydrofolate; rpiA involved in ribose metabolism; and protein genes with an unknown function (yhhQ and ydiK). The PurR site was shown to have different structure in different genomes. Thus, the tendency for a decline of the conservatism of site positions 2 and 15 was observed in genomes of bacteria belonging to the Pasteurellaceae and Vibrionaceae groups.

  12. 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.

  13. 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

  14. 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 PAGES

    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

  15. 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

  16. Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase.

    PubMed

    Hannibal, Luciana; Somasundaram, Ramasamy; Tejero, Jesús; Wilson, Adjele; Stuehr, Dennis J

    2011-11-11

    Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.

  17. 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.

  18. Purine biosynthesis in L1210 leukemia cells is inhibited by 7-hydroxymethotrexate (7-OH-MTX) polyglutamates (PGS)

    SciTech Connect

    Seither, R.L.; Matherly, L.H.; Goldman, I.D.

    1986-05-01

    The biochemical basis for 7-OH-MTX cytotoxicity was examined in L1210 tumor cells. Cells were exposed to 100 ..mu..M 7-OH-MTX (approx. 50% growth inhibition) or 10 ..mu..M methotrexate (MTX) (approx. 95% growth inhibition) for 6 hrs to allow high levels of PGS to accumulate. Dihydrofolate reductase (DHFR) activity was assessed by dihydrofolate (FH/sub 2/) pools labeled with 5-formyl-(/sup 3/H)-tetrahydrofolate (5..mu..M) or /sup 3/H-folic acid (1 ..mu..M). FH/sub 2/ was not elevated above control levels in 7-OH-MTX treated cells, in contrast to MTX treated cells in which FH/sub 2/ increased 4- to 7-fold. /sup 3/H-Deoxyuridine incorporation into DNA was not inhibited in cells containing high levels (11.5 nmol/g dry wt.) of 7-OH-MTX tetraglutamate (7-OH-4-NH/sub 2/-10-CH/sub 3/-PteGlu/sub 4/), well in excess of the DHFR-binding capacity (7.3 +/- 0.9 nmol/g), indicating a normal rate of thymidylate synthesis. Although small amounts of 7-OH-MTX and its PGS were bound to DHFR in L1210 cells, as assessed by gel filtration, there was evidence for the preferential binding of 7-OH-MTX tetraglutamate. In all cases this was well below the DHFR binding capacity, consistent with normal rates of deoxyuridine metabolism and FH/sub 2/ levels in the cell. Incorporation of /sup 14/C-formate (60 min) into thymidylate and amino acids was unaffected by 7-OH-MTX, yet incorporation into purines was inhibited over 50%, supporting a block(s) in de novo purine biosynthesis.

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

    PubMed

    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; Gregory, Jesse F

    2014-07-01

    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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. FORMATE—PYRUVATE EXCHANGE REACTION IN STREPTOCOCCUS FAECALIS II.

    PubMed Central

    Oster, M. O.; Wood, N. P.

    1964-01-01

    Oster, M. O. (A. & M. College of Texas, College Station), and N. P. Wood. Formate-pyruvate exchange reaction in Streptococcus faecalis. II. Reaction conditions for cell extracts. J. Bacteriol. 87:104–113. 1964.—In contrast to intact cells of Streptococcus faecalis, no stimulation of the formate-pyruvate exchange reaction was observed in cell extracts when yeast extract was added to the reaction mixture. A heated extract of Micrococcus lactilyticus, vitamin K5, ferrous sulfate, and ferrous ammonium sulfate stimulated an active exchange by protecting the system from oxygen. Tetrahydrofolate, 2,3-dimercaptopropanol, and sodium sulfide provided partial protection, whereas ascorbate, glutathione, sodium hydrosulfite, ammonium sulfide, and sodium bisulfite gave insufficient protection or were inhibitory. Oxidation-reduction (O-R) indicators were not inhibitory and were used to estimate the O-R potentials of reaction mixtures. A potential at least as negative as −125 mv was estimated to be necessary to preserve or initiate formate-pyruvate exchange activity. The reaction operated over a narrow pH range when strict anaerobic conditions were not maintained but, when the system was suitably poised, the pH range was broader. The influence of high phosphate concentrations was less under strictly anaerobic conditions, and orthophosphate could be replaced by small amounts of pyrophosphate. Effect of temperature, time, and amount of extract is presented. Addition of reduced benzyl viologen and hydrogen-saturated palladium in the buffer during 8 hr of dialysis prevented inactivation of extracts. Recovery of activity could be obtained after ammonium sulfate treatment when a combination of palladium chloride, neutral red, and hydrogen bubbling were used. PMID:14102842

  5. Decoding how a soil bacterium extracts building blocks and metabolic energy from ligninolysis provides road map for lignin valorization

    PubMed Central

    Varman, Arul M.; He, Lian; Follenfant, Rhiannon; Wu, Weihua; Wemmer, Sarah; Wrobel, Steven A.; Tang, Yinjie J.; Singh, Seema

    2016-01-01

    Sphingobium sp. SYK-6 is a soil bacterium boasting a well-studied ligninolytic pathway and the potential for development into a microbial chassis for lignin valorization. An improved understanding of its metabolism will help researchers in the engineering of SYK-6 for the production of value-added chemicals through lignin valorization. We used 13C-fingerprinting, 13C metabolic flux analysis (13C-MFA), and RNA-sequencing differential expression analysis to uncover the following metabolic traits: (i) SYK-6 prefers alkaline conditions, making it an efficient host for the consolidated bioprocessing of lignin, and it also lacks the ability to metabolize sugars or organic acids; (ii) the CO2 release (i.e., carbon loss) from the ligninolysis-based metabolism of SYK-6 is significantly greater than the CO2 release from the sugar-based metabolism of Escherichia coli; (iii) the vanillin catabolic pathway (which is the converging point of majority of the lignin catabolic pathways) is coupled with the tetrahydrofolate-dependent C1 pathway that is essential for the biosynthesis of serine, histidine, and methionine; (iv) catabolic end products of lignin (pyruvate and oxaloacetate) must enter the tricarboxylic acid (TCA) cycle first and then use phosphoenolpyruvate carboxykinase to initiate gluconeogenesis; and (v) 13C-MFA together with RNA-sequencing differential expression analysis establishes the vanillin catabolic pathway as the major contributor of NAD(P)H synthesis. Therefore, the vanillin catabolic pathway is essential for SYK-6 to obtain sufficient reducing equivalents for its healthy growth; cosubstrate experiments support this finding. This unique energy feature of SYK-6 is particularly interesting because most heterotrophs rely on the transhydrogenase, the TCA cycle, and the oxidative pentose phosphate pathway to obtain NADPH. PMID:27634497

  6. Hypoxia-resistant profile implies vulnerability of cancer stem cells to physiological agents, which suggests new therapeutic targets

    PubMed Central

    Cipolleschi, Maria Grazia; Marzi, Ilaria; Santini, Roberta; Fredducci, David; Vinci, Maria Cristina; D’Amico, Massimo; Rovida, Elisabetta; Stivarou, Theodora; Torre, Eugenio; Dello Sbarba, Persio; Stecca, Barbara; Olivotto, Massimo

    2014-01-01

    We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments. PMID:24200964

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  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. 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. The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases.

    PubMed

    Thillet, J; Adams, J A; Benkovic, S J

    1990-05-29

    A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Molecular basis of dimethylglycine dehydrogenase deficiency associated with pathogenic variant H109R

    PubMed Central

    McAndrew, R. P.; Vockley, J.

    2010-01-01

    Summary Dimethylglycine dehydrogenase (DMGDH) is a mitochondrial matrix flavoprotein that catalyses the demethylation of dimethylglycine to form sarcosine, accompanied by the reduction of the covalently bound FAD cofactor. Electron-transfer flavoprotein reoxidizes the reduced flavin and transfers reducing equivalents to the main mitochondrial respiratory chain through the enzyme ETF-ubiquinone oxidoreductase. DMGDH plays a prominent role in choline and 1-carbon metabolism. We have expressed the mature form of human DMGDH and the H109R variant identified in a DMGDH-deficient patient as N-terminally His6-tagged proteins in E. coli. The enzymes were purified to homogeneity by nickel affinity and anion exchange chromatography. The presence of FAD in the wild-type enzyme was confirmed by spectrophotometric analysis. The H109R variant, however, had only 47% of the wild-type level of bound flavin as expressed in E. coli, indicating its reduced affinity for FAD As previously described for rat enzyme studies, the wild-type human enzyme exhibited two Km values for N,N-dimethylglycine (Km1 = 0.039 ± 0.010 mmol/L and Km2 = 15.4 ± 1.2 mmol/L). The addition of 4 μmol/L tetrahydrofolate resulted in a slight decrease in specific activity and a substantial decrease in Km2 (1.10 ± 0.55 mmol/L). The flavinated H109R variant protein exhibited a 27-fold decrease in specific activity and a 65-fold increase in Km, explaining its pathogenicity. Additionally, the current expression system represents a significant improvement over a previously described rat DMGDH expression system and will enhance our ability to further study this important metabolic enzyme. PMID:18937046

  16. 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

  17. 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.

  18. 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

  19. Serine hydroxymethyltransferase from Escherichia coli: purification and properties.

    PubMed Central

    Schirch, V; Hopkins, S; Villar, E; Angelaccio, S

    1985-01-01

    Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M. Plamann, L. Stauffer, M. Urbanowski, and G. Stauffer, Nucleic Acids Res. 11:2065-2074, 1983). The enzyme contained no disulfide bonds but had one sulfhydryl group on the surface of the protein. Several sulfhydryl reagents reacted with this exposed group and inactivated the enzyme. Spectra of the enzyme in the presence of substrates and substrate analogs showed that the enzyme formed the same complexes and in similar relative concentrations as previously observed with the cytosolic and mitochondrial rabbit liver isoenzymes. Kinetic studies with substrates showed that the affinity and synergistic binding of the amino acid and folate substrates were similar to those obtained with the rabbit liver isoenzymes. The enzyme catalyzed the cleavage of threonine, allothreonine, and 3-phenylserine to glycine and the corresponding aldehyde in the absence of tetrahydrofolate. The enzyme was also inactivated by D-alanine caused by the transamination of the active site pyridoxal phosphate to pyridoxamine phosphate. This substrate specificity was also observed with the rabbit liver isoenzymes. We conclude that the reaction mechanism and the active site structure of E. coli serine hydroxymethyltransferase are very similar to the mechanism and structure of the rabbit liver isoenzymes. PMID:3891721

  20. [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

  1. 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.

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

    PubMed

    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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Biochemical fossils of the ancient transition from geoenergetics to bioenergetics in prokaryotic one carbon compound metabolism.

    PubMed

    Sousa, Filipa L; Martin, William F

    2014-07-01

    The deep dichotomy of archaea and bacteria is evident in many basic traits including ribosomal protein composition, membrane lipid synthesis, cell wall constituents, and flagellar composition. Here we explore that deep dichotomy further by examining the distribution of genes for the synthesis of the central carriers of one carbon units, tetrahydrofolate (H4F) and tetrahydromethanopterin (H4MPT), in bacteria and archaea. The enzymes underlying those distinct biosynthetic routes are broadly unrelated across the bacterial-archaeal divide, indicating that the corresponding pathways arose independently. That deep divergence in one carbon metabolism is mirrored in the structurally unrelated enzymes and different organic cofactors that methanogens (archaea) and acetogens (bacteria) use to perform methyl synthesis in their H4F- and H4MPT-dependent versions, respectively, of the acetyl-CoA pathway. By contrast, acetyl synthesis in the acetyl-CoA pathway - from a methyl group, CO2 and reduced ferredoxin - is simpler, uniform and conserved across acetogens and methanogens, and involves only transition metals as catalysts. The data suggest that the acetyl-CoA pathway, while being the most ancient of known CO2 assimilation pathways, reflects two phases in early evolution: an ancient phase in a geochemically confined and non-free-living universal common ancestor, in which acetyl thioester synthesis proceeded spontaneously with the help of geochemically supplied methyl groups, and a later phase that reflects the primordial divergence of the bacterial and archaeal stem groups, which independently invented genetically-encoded means to synthesize methyl groups via enzymatic reactions. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  9. Determination of folates by HPLC-chemiluminescence using a ruthenium(II)-cerium(IV) system, and its application to pharmaceutical preparations and supplements.

    PubMed

    Ikeda, Rie; Ichiyama, Kosuke; Tabuchi, Naoto; Wada, Mitsuhiro; Kuroda, Naotaka; Nakashima, Kenichiro

    2014-11-01

    A chemiluminescence (CL) reaction of folic acid (FA) with ruthenium (II) and cerium (IV) was applied to quantify FA-related compounds such as FA, dihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid and methotrexate (MTX). Among the FAs, 5-methyltetrahydrofolic acid provided the highest CL intensity. HPLC-CL detection of FA was applied to quantify FA in pharmaceutical preparations and supplements. Analytical samples were separated on a semi-micro ODS column with a mixture of 20 mM phosphate buffer (pH 5.7) and acetonitrile (94 : 6, v/v %). The separated samples were mixed with a post-column CL reagent consisting of 1.5 mM Ru(bipy)3 (2+) and 1.0 mM Ce(SO4)2 , then the generated CL was monitored. The calibration range for FA was 10-100 μM and the limit of detection was 1.34 μM (signal-to-noise ratio of 3). Repeatabilities were 4.2, 4.6 and 5.0 RSD% (10, 25, 50 μM), and the recoveries for FA supplement, vitamin B complex supplement and FA-containing medication (tablet) were 102.4 ± 10.5, 103.3 ± 13.3 and 100.3 ± 8.5%, respectively. The described method is robust against changes in the chromatographic parameters of ± 3.3 or ± 1.5%. The measured FA content corresponded well to the labeled content of FA-containing products (100.6-104.9%), demonstrating the precision and accuracy of this method for the evaluation of FA pharmaceutical preparations.

  10. Nitrous oxide inactivation of cobalamin-dependent methionine synthase from Escherichia coli: characterization of the damage to the enzyme and prosthetic group.

    PubMed

    Drummond, J T; Matthews, R G

    1994-03-29

    Nitrous oxide, or laughing gas, is an anaesthetic agent that inactivates cobalamin-dependent methionine synthase. This enzyme uses the highly reactive, enzyme-bound cob(I)alamin oxidation state of the prosthetic group to effect methyl group transfer from 5-methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. The cob(I)alamin is capable of reductively degrading nitrous oxide, and here we characterize the modifications that occur to the Escherichia coli enzyme following electrochemical inactivation. Methionine synthase was inactivated on a milligram scale by equilibrating enzyme containing bound cob(II)alamin with a reduced electrochemical mediator to give the reactive cob(I)alamin state under an anaerobic atmosphere of nitrous oxide. The primary damage occurs to a 37.2-kDa domain that binds S-adenosylmethionine (AdoMet), and inactive enzyme can no longer be reductively methylated using AdoMet. The damage is oxidative, and it includes the covalent addition of the mediator, triquat, to the enzyme selectively at valine 1177, as well as the formation of a covalent cross-link between peptides containing the only two cysteines within this domain. Spectrally, the prosthetic group bound to inactive enzyme resembles cob(II)alamin, although some loss in absorbance is apparent. When the enzyme was reconstituted with [57Co]cobalamin and the inactivation repeated, the cobalamin was recovered unmodified in approximately 75% yield, but two products derived from the cobalamin were also observed. We interpret the finding of oxidatively modified products as strong evidence that reductive degradation of nitrous oxide releases a potent oxidant, presumably hydroxyl radical or its equivalent, that is capable of modifying sites proximal to the cobalamin. PMID:8142374

  11. 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

  12. 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.

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

    PubMed

    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.

  14. Significant association of methylenetetrahydrofolate reductase single nucleotide polymorphisms with prostate cancer susceptibility in taiwan.

    PubMed

    Wu, Hsi-Chin; Chang, Chao-Hsiang; Tsai, Ru-Yin; Lin, Chih-Hsueh; Wang, Rou-Fen; Tsai, Chia-Wen; Chen, Kuen-Bao; Yao, Chun-Hsu; Chiu, Chang-Fang; Bau, Da-Tian; Lin, Cheng-Chieh

    2010-09-01

    Prostate cancer is the most common cause of cancer death in men and is a major health problem worldwide. Methylene tetrahydrofolate reductase (MTHFR) plays an important role in folate metabolism and is also an important source of DNA methylation and DNA synthesis (nucleotide synthesis). To assess the association and interaction of genotypic polymorphisms in MTHFR and lifestyle factors with prostate cancer in Taiwan, we investigated two well-known polymorphic variants of MTHFR, C677T (rs1801133) and A1298C (rs1801131), analyzed the association of specific genotypes with prostate cancer susceptibility, and discussed their joint effects with individual habits on prostate cancer risk. In total, 218 patients with prostate cancer and 436 healthy controls recruited from the China Medical Hospital in central Taiwan were genotyped for these polymorphisms with prostate cancer susceptibility. We found the MTHFR C677T but not the A1298C genotype was differently distributed between the prostate cancer and control groups. The T allele of MTHFR C677T conferred a significantly (p=0.0011) decreased risk of prostate cancer. As for the A1298C polymorphism, there was no difference in distribution between the prostate cancer and control groups. Gene interactions with smoking were significant for MTHFR C677T polymorphism. The MTHFR C677T CT and TT genotypes in association with smoking conferred a decreased risk of 0.501 (95% confidence interval=0.344-0.731) for prostate cancer. Our results provide the first evidence that the C allele of MTHFR C677T may be associated with the development of prostate cancer and may be a novel useful marker for primary prevention and anticancer intervention.

  15. Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii.

    PubMed Central

    Comley, J C; Mullin, R J; Wolfe, L A; Hanlon, M H; Ferone, R

    1991-01-01

    Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii. PMID:1759815

  16. Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

    PubMed

    Chen, Shengxi; Fahmi, Nour Eddine; Bhattacharya, Chandrabali; Wang, Lin; Jin, Yuguang; Benkovic, Stephen J; Hecht, Sidney M

    2013-11-26

    In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational

  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. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Frequency and association of 1691 (G>A) FVL, 20210 (G>A) PT and 677 (C>T) MTHFR with deep vein thrombosis in the population of Bosnia and Herzegovina

    PubMed Central

    Terzić, R; Jerkić, Z; Avdić, A; Pođanin, M

    2016-01-01

    Abstract The 1691 (G>A) factor V Leiden (FVL) and 20210 (G>A) prothrombin (PT) mutations are the two most common genetic risk factors in venous thromboembolism. The 677 (C>T) methylene tetrahydrofolate reductase (MTHFR) mutation is the most frequently mentioned as an independent genetic risk factor for venous thromboembolism. As there are limited published data on the prevalence of the 1691, 20210 and 677 mutations in our population, the aim of this study was to determine the frequencies and association of these deep vein thrombosis mutations in the Bosnian population. This study included 111 thromboembolic patients and 207 healthy subjects with absence of known risk factors for venous thromboembolism. Genotyping of the 1691, 20210 and 677 mutations was done by polymerase chain reaction (PCR), followed by restriction digestion with MnlI, HindIII and HinfI enzymes. Out of the 111 patients, 18.0% were heterozygous and 2.70% were homozygous for the 1691 mutation. Among 207 healthy controls, 3.86%, were heterozygous for the 1691 mutation. This study confirmed the association of the 1691 mutation with deep vein thrombosis in the Bosnian population odds ratio (OR) [95% confidence interval (CI)] = 6.0 (2.62-14.14); p = 0.0001). The 20210 mutation was detected in 2.70% of patients and it was totally absent in the control group. Allele and genotype frequency of 677 did not differ significantly between the cases and controls (χ2 = 1.03; p = 0.309). PMID:27785407

  6. 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.

  7. Folate metabolic pathways in Leishmania.

    PubMed

    Vickers, Tim J; Beverley, Stephen M

    2011-01-01

    Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compounds developed originally for treatment of human cancers or other infectious agents.

  8. Concentrations of unmetabolized folic acid and primary folate forms in plasma after folic acid treatment in older adults.

    PubMed

    Obeid, Rima; Kirsch, Susanne H; Kasoha, Mariz; Eckert, Rudolf; Herrmann, Wolfgang

    2011-05-01

    Folate deficiency can cause age-related disease. Folic acid (FA) has been used in studies aiming at disease prevention. Recently, unmetabolized FA in plasma raised public health concerns; but numerous studies used FA for disease prevention. Concentrations of the folate forms FA, 5-methyltetrahydrofolate (5-MTHF), and tetrahydrofolate (THF) were measured before and after 3-week placebo or FA 5 mg, vitamin B6 40 mg, and cyanocobalamin 2 mg per day administrated to 74 older adults (median age, 82 years). Concentrations of 5-MTHF and total homocysteine (tHcy) (r = -0.392) and S-adenosylmethionine (r = 0.329) were correlated at baseline. Twenty-six percent of the elderly subjects had unmetabolized FA in plasma at the start, and concentrations of FA were increased after 3 weeks of FA treatment (median FA = 0.08 nmol/L at baseline and 15.3 nmol/L at the end of the treatment in the vitamin group). Folic acid caused a 10- and a 5-fold increase in 5-MTHF and THF, respectively, and lowered tHcy (median tHcy = 17.2 μmol/L at baseline vs 9.0 μmol/L after treatment). Concentrations of unmetabolized FA were positively related to those of 5-MTHF and THF. People showed wide variations in folate forms at baseline, but these were reduced after FA treatment. Folic acid given to older adults is mostly converted to THF and 5-MTHF and lowered concentrations of tHcy, but caused a substantial increase in unmetabolized FA in the plasma.

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

    PubMed

    Kalmbach, Renee D; Choumenkovitch, Silvina F; Troen, Aron P; Jacques, Paul F; D'Agostino, Ralph; Selhub, Jacob

    2008-12-01

    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. The functional impact of this polymorphism has not yet been demonstrated. The objective of this research was to determine the effects of the DHFR mutation with respect to folate status and assess influence of folic acid intake on these relations. The relationship between DHFR genotype and plasma concentrations of circulating folic acid, total folate, total homocysteine, and concentrations of RBC folate was determined in 1215 subjects from the Framingham Offspring Study. There was a significant interaction between DHFR genotype and folic acid intake with respect to the prevalence of high circulating unmetabolized folic acid (defined as >85th percentile). Folic acid intake of >or=500 microg/d increased the prevalence of high circulating unmetabolized folic acid in subjects with the deletion (del/del genotype (47.0%) compared with the wild type (WT)/del (21.4%) and wild type (WT)/WT genotypes (24.4%) (P for interaction = 0.03). Interaction between the DHFR polymorphism and folic acid intake was also seen with respect to RBC folate (P for interaction = 0.01). When folic acid intake was <250 microg/d, the del/del genotype was associated with significantly lower RBC folate (732.3 nmol/L) compared with the WT/WT genotype (844.4 nmol/L). Our results suggest the del/del polymorphism in DHFR is a functional polymorphism, because it limits assimilation of folic acid into cellular folate stores at high and low folic acid intakes.

  10. Is 5-methyltetrahydrofolate an alternative to folic acid for the prevention of neural tube defects?

    PubMed

    Obeid, Rima; Holzgreve, Wolfgang; Pietrzik, Klaus

    2013-09-01

    Women have higher requirements for folate during pregnancy. An optimal folate status must be achieved before conception and in the first trimester when the neural tube closes. Low maternal folate status is causally related to neural tube defects (NTDs). Many NTDs can be prevented by increasing maternal folate intake in the preconceptional period. Dietary folate is protective, but recommending increasing folate intake is ineffective on a population level particularly during periods of high demands. This is because the recommendations are often not followed or because the bioavailability of food folate is variable. Supplemental folate [folic acid (FA) or 5-methyltetrahydrofolate (5-methylTHF)] can effectively increase folate concentrations to the level that is considered to be protective. FA is a synthetic compound that has no biological functions unless it is reduced to dihydrofolate and tetrahydrofolate. Unmetabolized FA appears in the circulation at doses of >200 μg. Individuals show wide variations in their ability to reduce FA. Carriers of certain polymorphisms in genes related to folate metabolism or absorption can better benefit from 5-methylTHF instead of FA. 5-MethylTHF [also known as (6S)-5-methylTHF] is the predominant natural form that is readily available for transport and metabolism. In contrast to FA, 5-methylTHF has no tolerable upper intake level and does not mask vitamin B12 deficiency. Supplementation of the natural form, 5-methylTHF, is a better alternative to supplementation of FA, especially in countries not applying a fortification program. Supplemental 5-methylTHF can effectively improve folate biomarkers in young women in early pregnancy in order to prevent NTDs.

  11. Isolation and characterization of two new homoacetogenic hydrogen-utilizing bacteria from the human intestinal tract that are closely related to Clostridium coccoides.

    PubMed Central

    Kamlage, B; Gruhl, B; Blaut, M

    1997-01-01

    Two gram-positive, strictly anoxic, coccoid- to rod-shaped strains of bacteria, Clostridium coccoides 1410 and C. coccoides 3110, were isolated from human feces on the typical homoacetogenic substrates formate plus H2 plus CO2 (strain 1410) and vanillate plus H2 plus CO2 (strain 3110) in the presence of 2-bromoethanesulfonate to inhibit methanogenesis. On the basis of 16S rRNA sequencing, DNA-DNA hybridization, and physiological and morphological parameters, both isolates are closely related to C. coccoides DSM 935T. The G+C contents of the DNA were 46.1 and 46.2 mol% for C. coccoides 1410 and C. coccoides 3110, respectively. Cytochromes could not be detected. Formate was degraded exclusively to acetate, whereas vanillate was O-demethylated, resulting in acetate and 3,4-dihydroxybenzoate, the latter being further decarboxylated to catechol. In the presence of organic substrates, H2 was cometabolized to acetate, but both strains failed to grow autotrophically. Lactose, lactulose, sorbitol, glucose, and various other carbohydrates supported growth as well. Untypical of homoacetogens, glucose and sorbitol were fermented not exclusively to acetate; instead, considerable amounts of succinate and D-lactate were produced. H2 was evolved from carbohydrates only in negligible traces. Acetogenesis from formate plus H2 plus CO2 or vanillate plus H2 plus CO2 was constitutive, whereas utilization of carbohydrates was inducible. Hydrogenase, CO dehydrogenase, formate dehydrogenase, and all of the tetrahydrofolic acid-dependent, C1 compound-converting enzymes of the acetyl-coenzyme A pathway of homoacetogenesis were present in cell extracts. PMID:9143110

  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

    Nayak, Dipti D; Marx, Christopher J

    2014-12-01

    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.

  13. Intestinal Folate Absorption

    PubMed Central

    Olinger, Edward J.; Bertino, Joseph R.; Binder, Henry J.

    1973-01-01

    These studies were designed to determine whether pteroylmonoglutamic acid (PGA) at physiologic concentrations is transported across the small intestine unaltered or is reduced and methylated to the circulating folate form (5-methyltetrahydrofolate [5-MeFH4]) during absorption. [3H]PGA was incubated in vitro on the mucosal side of rat jejunum. Of the folate transferred to the serosal side, the percent identified as 5-MeFH4 by DEAE-Sephadex chromtography was inversely related to the initial mucosa PGA concentration: at 7, 20, and 2,000 nM, 44%, 34%, and 2%, respectively, was converted to 5-MeFH4. In contrast, less than 4% of the folate transferred across ileal mucosa was 5-MeFH4 when the initial mucosa concentration was 20 nM. Specific activity of dihydrofolate (DHF) reductase, the enzyme responsible for converting PGA to tetrahydrofolic acid, was measured in villus homogenates and was significantly greater in the jejunum than in the ileum. 1,000 nM methotrexate (MTX), a DHF reductase inhibitor, markedly inhibited PGA conversion to 5-MeFH4 by the jejunum. Studies of transmural flux, initial rate of mucosal entry (influx) and mucosal accumulation (uptake) of folate were also performed. Although MTX did not alter the influx of PGA, MTX decreased jejunal mucosal uptake but increased transmural movement. Transmural folate movement across ileal mucosa was greater than across jejunal mucosa although mucosal uptake was greater in the jejunum than in the ileum. These results could explain previous studies which have failed to identify conversion of PGA to 5-MeFH4 when intestinal preparations have been exposed to higher and less physiologic concentrations of PGA. Further, these studies suggest that 5-MeFH4 may be retained by the jejunal mucosa. PMID:4727453

  14. Cofactor-Mediated Conformational Dynamics Promote Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway

    PubMed Central

    2016-01-01

    The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP+). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP+ and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants. PMID:26147643

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum.

    PubMed

    Paarup, Maiken; Friedrich, Michael W; Tindall, Brian J; Finster, Kai

    2006-01-01

    A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)*

    PubMed Central

    Yamagami, Ryota; Yamashita, Koki; Nishimasu, Hiroshi; Tomikawa, Chie; Ochi, Anna; Iwashita, Chikako; Hirata, Akira; Ishitani, Ryuichiro; Nureki, Osamu; Hori, Hiroyuki

    2012-01-01

    The conserved U54 in tRNA is often modified to 5-methyluridine (m5U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m5U54 is produced by folate/FAD-dependent tRNA (m5U54) methyltransferase (TrmFO). TrmFO utilizes N5,N10-methylenetetrahydrofolate (CH2THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [14C]CH2THF was supplied from [14C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m1A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m5U54, m1A58, and s2U54 modifications on m5s2U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme. PMID:23095745

  9. 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

  10. Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis.

    PubMed

    Witt, Teah L; Matherly, Larry H

    2002-12-23

    Site-directed mutagenesis was used to characterize the functional role of lysine-411, a conserved amino acid located in putative transmembrane domain (TMD) 11 of the human reduced folate carrier (hRFC). Lysine-411 was mutagenized to arginine, glutamate, and leucine, and the mutant constructs (K411R-, K411E-, and K411L-hRFC, respectively) were transfected into hRFC-deficient K562 cells. The mutant hRFC constructs were all expressed at high levels and restored 22-36% of the methotrexate (MTX) transport level in wild-type (K43-6) hRFC transfectants. Although 5-formyl tetrahydrofolate (5-CHO-H(4)PteGlu) uptake levels for both the K411E- and K411L-hRFCs were also impaired (approximately 33% and 28%, respectively), a complete restoration of the wild-type level was observed for K411R-hRFC. While loss of MTX transport activity for the K411R-hRFC transfectant was associated with an incomplete restoration of MTX sensitivity compared to K43-6 cells, these cells were similarly sensitive to Tomudex. The K411R-hRFC transfectants showed an approximately threefold decreased growth requirement for 5-CHO-H(4)PteGlu compared to K43-6 cells. The 5-CHO-H(4)PteGlu transport stimulation observed for the wild-type carrier in chloride-free buffer was also observed for K411R-hRFC, however, this response was decreased for the K411E- and K411L-hRFCs. The preservation of low levels of transport for the K411E- and K411L-hRFCs suggest that the amino acid at position 411 does not directly participate in the binding of anionic hRFC substrates. However, a functionally important role for a basic amino acid at position 411 was, nonetheless, implied by the increased MTX transport for wild-type hRFC over the K411 mutant hRFCs, and the highly selective uptake of 5-CHO-H(4)PteGlu over MTX for K411R-hRFC. PMID:12488038

  11. [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

  12. Loss of function of folylpolyglutamate synthetase 1 reduces lignin content and improves cell wall digestibility in Arabidopsis

    DOE PAGES

    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

  13. Folic acid handling by the human gut: implications for food fortification and supplementation123

    PubMed Central

    Patanwala, Imran; King, Maria J; Barrett, David A; Rose, John; Jackson, Ralph; Hudson, Mark; Philo, Mark; Dainty, Jack R; Wright, Anthony JA; Finglas, Paul M; Jones, David E

    2014-01-01

    Background: Current thinking, which is based mainly on rodent studies, is that physiologic doses of folic acid (pterylmonoglutamic acid), such as dietary vitamin folates, are biotransformed in the intestinal mucosa and transferred to the portal vein as the natural circulating plasma folate, 5-methyltetrahydrofolic acid (5-MTHF) before entering the liver and the wider systemic blood supply. Objective: We tested the assumption that, in humans, folic acid is biotransformed (reduced and methylated) to 5-MTHF in the intestinal mucosa. Design: We conducted a crossover study in which we sampled portal and peripheral veins for labeled folate concentrations after oral ingestion with physiologic doses of stable-isotope–labeled folic acid or the reduced folate 5-formyltetrahydrofolic acid (5-FormylTHF) in 6 subjects with a transjugular intrahepatic porto systemic shunt (TIPSS) in situ. The TIPSS allowed blood samples to be taken from the portal vein. Results: Fifteen minutes after a dose of folic acid, 80 ± 12% of labeled folate in the hepatic portal vein was unmodified folic acid. In contrast, after a dose of labeled 5-FormylTHF, only 4 ± 18% of labeled folate in the portal vein was unmodified 5-FormylTHF, and the rest had been converted to 5-MTHF after 15 min (postdose). Conclusions: The human gut appears to have a very efficient capacity to convert reduced dietary folates to 5-MTHF but limited ability to reduce folic acid. Therefore, large amounts of unmodified folic acid in the portal vein are probably attributable to an extremely limited mucosal cell dihydrofolate reductase (DHFR) capacity that is necessary to produce tetrahydrofolic acid before sequential methylation to 5-MTHF. This process would suggest that humans are reliant on the liver for folic acid reduction even though it has a low and highly variable DHFR activity. Therefore, chronic liver exposure to folic acid in humans may induce saturation, which would possibly explain reports of systemic circulation of

  14. A New Role for the Ankyrin Repeat Revealed by the Study of the N-formyltransferase from Providencia alcalifaciens

    PubMed Central

    Woodford, Colin R.; Thoden, James B.; Holden, Hazel M.

    2015-01-01

    N -formylated sugars such as 3,6-dideoxy-3-formamido-D-glucose (Qui3NFo) have been observed on the lipopolysaccharides of various pathogenic bacteria including Providencia alcalifaciens, a known cause of gastroenteritis. These unusual carbohydrates are synthesized in vivo as dTDP-linked sugars. The biosynthetic pathway for the production of dTDP-Qui3NFo requires five enzymes with the last step catalyzed by an N-formyltransferase that utilizes N10-tetrahydrofolate as a cofactor. Here we describe a structural and functional investigation of the P. alcalifaciens N-formyltransferase, hereafter referred to as QdtF. For this analysis the structure of the dimeric enzyme was solved in the presence of N5-formyltetrahydrofolate, a stable cofactor, and dTDP-3,6-dideoxy-3-amino-D-glucose (dTDP-Qui3N) to 1.5-Å resolution. The overall fold of the subunit consists of three regions with the N-terminal and middle motifs followed by an ankyrin repeat domain. Whereas the ankyrin repeat is a common eukaryotic motif involved in protein:protein interactions, reports of its presence in prokaryotic enzymes have been limited. Unexpectedly, this ankyrin repeat houses a second binding pocket for dTDP-Qui3N, which is characterized by extensive interactions between the protein and the ligand. To address the effects of this second binding site on catalysis, a site-directed mutant protein, W305A, was constructed. Kinetic analyses demonstrated that the catalytic activity of the W305A variant was reduced by approximately sevenfold. The structure of the W305A mutant protein in complex with N5-formyltetrahydrofolate and dTDP-Qui3N was subsequently determined to 1.5-Å resolution. The electron density map clearly showed that ligand binding had been completely abolished in the auxiliary pocket. The wild-type enzyme was also tested for activity against dTDP-3,6-dideoxy-3-amino-D-galactose (dTDP-Fuc3N) as a substrate. Strikingly, sigmoidal kinetics were observed indicating homotropic allosteric

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

    PubMed

    Wang, Jian-Wei; Peng, Shu-You; Li, Jiang-Tao; Wang, Yong; Zhang, Zhi-Ping; Cheng, Yan; Cheng, De-Qing; Weng, Wei-Hong; Wu, Xiang-Song; Fei, Xiao-Zhou; Quan, Zhi-Wei; Li, Ji-Yu; Li, Song-Gang; Liu, Ying-Bin

    2009-08-18

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

  16. Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

    PubMed Central

    Baggott, J E; Vaughn, W H; Hudson, B B

    1986-01-01

    producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively. PMID:2431676

  17. Association of seven functional polymorphisms of one-carbon metabolic pathway with total plasma homocysteine levels and susceptibility to Parkinson's disease among South Indians.

    PubMed

    Kumudini, Nadella; Uma, Addepally; Naushad, Shaik Mohammad; Mridula, Rukmini; Borgohain, Rupam; Kutala, Vijay Kumar

    2014-05-01

    This study from South India was performed to ascertain the impact of seven functional polymorphisms of one-carbon metabolic pathway on total plasma homocysteine levels and susceptibility to PD. A total of 151 cases of Parkinson's disease and 416 healthy controls were analyzed for fasting plasma homocysteine levels by reverse phase HPLC. PCR-RFLP approaches were used to analyze glutamate carboxypeptidase II (GCPII) 1561 C>T, reduced folate carrier 1 (RFC1) 80 G>A, cytosolic serine hydroxymethyl transferase (cSHMT) 1420 C>T, methylene tetrahydrofolate reductase (MTHFR) 677 C>T, methionine synthase (MTR) 2756 A>G and methionine synthase reductase (MTRR) 66 A>G polymorphisms. PCR-AFLP was used for the analysis of thymidylate synthase (TYMS) 5'-UTR 28bp tandem repeat. PD cases exhibited elevated plasma homocysteine levels compared to controls (men: 28.8 ± 6.9 vs. 16.4 ± 8.8 μmol/L; women: 25.4 ± 5.3 vs. 11.2 ± 5.1μmol/L). Homocysteine levels showed positive correlation with male gender (r=0.39, p<0.0001) and MTRR 66 A>G (r=0.31, p<0.0001) whereas an inverse correlation was observed with cSHMT 1420 C>T polymorphism. MTRR 66 A>G polymorphism showed independent risk for PD (OR: 3.42, 95% CI: 2.35-4.98) whereas cSHMT 1420 C>T conferred protection against PD (OR: 0.11, 95% CI: 0.07-0.17). Multifactor dimensionality reduction analysis showed synergistic interactions between MTHFR 677 C>T and MTRR 66 A>G, whereas cSHMT 1420 C>T exhibited counteracting interactions in altering susceptibility to PD. To conclude, PD cases exhibited hyperhomocysteinemia and MTRR 66 A>G and cSHMT 1420 C>T gene variants were shown to modulate PD risk by altering the homocysteine levels.

  18. Characterization of the gallate dioxygenase gene: three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6.

    PubMed

    Kasai, Daisuke; Masai, Eiji; Miyauchi, Keisuke; Katayama, Yoshihiro; Fukuda, Masao

    2005-08-01

    Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase beta and alpha subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K(m) for gallate and the V(max) were determined to be 66.9 +/- 9.3 microM and 42.7 +/- 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

  19. 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

  20. 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

  1. 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

  2. Effects of folic acid and vitamin B12 supplements on folate and homocysteine metabolism in pigs during early pregnancy.

    PubMed

    Guay, Frédéric; Jacques Matte, J; Girard, Christiane L; Palin, Marie-France; Giguère, Alain; Laforest, Jean-Paul

    2002-09-01

    The present experiment aimed to determine the effects of supplements of folic acid (FA) alone or in combination with vitamin B12 on folate and homocysteine metabolism in gestating nulliparous Yorkshire-Landrace (YL) and multiparous Landrace (LD) occidental sows and multiparous Chinese Meishan-Landrace (ML) sows. LD sows were randomly assigned to two treatments: 0 or 15 mg FA/kg diet while YL and ML sows were assigned to three treatments: 0 mg FA/kg diet, 15 mg FA/kg or 15 mg vitamin B12/kg diet. Supplements were given from the oestrus preceding insemination up to slaughter on day 15 of gestation. At slaughter, a uterine flush was collected to determine uterine contents of homocysteine, methionine, tetrahydrofolate (THF), 5-methyl-THF, pyridoxal 5-phosphate (P5P) and vitamin B12. Blood samples were taken at first oestrus, at insemination and on days 5, 10 and 15 of gestation to determine plasma concentrations of homocysteine, methionine, THF, 5-methyl-THF, P5P, vitamin B12 and relative total folate-binding capacity. In occidental sows (YL and LD), the FA supplement tended to decrease uterine flush content of homocysteine (P=0.06) and concentrations of plasma homocysteine (P=0.09). Nulliparous YL sows had lower concentrations of plasma homocysteine, methionine, THF and 5-methyl-THF (P<0.05) than multiparous LD sows. Multiparous ML and LD sows had similar concentrations of plasma THF, 5-methyl-THF, methionine and vitamin B12, but ML sows had lower concentrations of plasma homocysteine (P<0.05). The vitamin B12 supplement increased concentrations of plasma vitamin B12 (P<0.05) both in multiparous ML and nulliparous YL sows, but had no effect on the composition of either uterine flush or plasma. The present results showed also that sows had a low vitamin B12 status (<200 pg/ml) and high circulating homocysteine levels (>15 microm) during the first 15 d of gestation. Furthermore, the vitamin B12 content in uterine secretions represented between 180 and 300 % of the total

  3. Fibromyalgia, mood disorders, and intense creative energy: A1AT polymorphisms are not always silent.

    PubMed

    Schmechel, Donald E; Edwards, Christopher L

    2012-12-01

    Persons with single copies of common alpha-1-antitrypsin polymorphisms such as S and Z are often considered "silent carriers". Published evidence however supports a complex behavioral phenotype or trait - intense creative energy ("ICE")-associated with A1AT polymorphisms. We now confirm that phenotype and present an association of fibromyalgia syndrome (FMS) and A1AT in a consecutive series of neurological patients. This is a retrospective case control series of 3176 consecutive patients presenting to Duke University Memory Clinic (747 patients) and to regional community-based Caldwell Hospital Neurology and Memory center (2429 patients). Work-up included medical history and examination, psychological evaluation, and genetic analysis. Chronic widespread pain (CWP) or FMS were diagnosed according to clinical guidelines, mostly as secondary diagnoses. Neurological patients carrying A1AT polymorphisms were common (ca 16% prevalence) and carriers had significantly higher use of inhaler and anxiolytic medications. Patients with ICE phenotype had a significantly higher proportion of A1AT polymorphisms (42%) compared to non-ICE patients (13%). Presence of CWP or FMS was common (14-22%) with average age at presentation of 56 years old and mostly female gender (82%). Patients with CWP/FMS had again significantly higher proportion of A1AT polymorphisms (38%) compared to other neurological patients (13%). Patients with anxiety disorders, bipolar I or bipolar II disorders or PTSD also had increased proportion of A1AT polymorphisms and significant overlap with ICE and FMS phenotype. Significant reductions in CWP/FMS prevalence are seen in apolipoprotein E4 carriers and methylene tetrahydrofolate reductase (MTHFR) mutation homozygotes. Since ICE phenotype is reported as a lifelong behavioral attribute, the presumption is that A1AT carriers have fundamental differences in brain development and inflammatory response. In support of this concept is finding those persons reporting a

  4. 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

  5. Pharmacokinetics of methanol and formate in female cynomolgus monkeys exposed to methanol vapors.

    PubMed

    Medinsky, M A; Dorman, D C; Bond, J A; Moss, O R; Janszen, D B; Everitt, J I

    1997-06-01

    metabolism of formate in primates depends on the availability of tetrahydrofolate, the same four monkeys were next placed on a folate-deficient diet until folate concentrations in red blood cells consistent with moderate folate deficiency (29 to 107 ng/mL) were achieved. Monkeys were then reexposed to the highest exposure concentration, 900 ppm 14C-methanol, for a similar 2-hour period, and again the pharmacokinetic data described above were obtained. Even with a reduced folate status, monkeys exposed to 900 ppm methanol for 2 hours had peak concentrations of methanol-derived formate that were well below the endogenous levels of formate. Although these results represent only a single exposure and therefore preclude broad generalizations, they do suggest the body contains sufficient folate stores to effectively detoxify small doses of methanol-derived formate from exogenous sources, such as those that might occur during normal use of automotive fuel.