scholarly journals Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria

2007 ◽  
Vol 189 (23) ◽  
pp. 8537-8545 ◽  
Author(s):  
Damien Leduc ◽  
Frédéric Escartin ◽  
H. Frederik Nijhout ◽  
Michael C. Reed ◽  
Ursula Liebl ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Thymidylate Synthase ◽  
Insertional Mutagenesis ◽  
Rhodobacter Capsulatus ◽  
De Novo ◽  
Reductase Activity ◽  
Direct Proof ◽  
Cellular Functions ◽  
Thymidylate Synthesis

ABSTRACT Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised.

scholarly journals Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

2020 ◽  
Author(s):  
Sharon Spizzichino ◽  
Dalila Boi ◽  
Giovanna Boumis ◽  
Roberta Lucchi ◽  
Francesca R. Liberati ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Protein Interactions ◽  
De Novo ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Aggregation State ◽  
Entire Complex ◽  
The Individual ◽  
Thymidylate Synthesis

ABSTRACTDe novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: thymidylate synthase (TYMS), serine hydroxymethyltransferase (SHMT) and dihydrofolate reductase (DHFR), targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, provide a useful tool to develop drugs targeting the entire complex instead of the individual components.

scholarly journals In SilicoScreening, Synthesis andIn VitroEvaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

2009 ◽  
Vol 6 (s1) ◽  
pp. S97-S102 ◽  
Author(s):  
A. G. Nerkar ◽  
A. K. Saxena ◽  
S. A. Ghone ◽  
A. K. Thaker
Keyword(s):  
Anticancer Activity ◽  
Dihydrofolate Reductase ◽  
De Novo ◽  
Human Cancer ◽  
Microwave Assisted Synthesis ◽  
Human Cancer Cell Lines ◽  
Reductase Inhibitors ◽  
Base Derivative ◽  
Dhfr Inhibitors

Dihydrofolate reductase (DHFR) is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report thein silicoscreening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino) quinazolinones (Quinazolinone Shiff's bases) QSB1-5and pyridine-4-carbohydrazide Shiff's bases (ISB1-5) derivatives and theirin vitroanticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and1H-NMR. These compounds were then subjected forin vitroanticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX) was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3(4-(N, N-dimethyl-amino)-phenyl) Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used inin vitroanticancer assay as per the NCI (National Cancer Institute) guidelines.

Effects of tobacco smoke on IL-16 in CD8+ cells from human airways and blood: a key role for oxygen free radicals?

2011 ◽  
Vol 300 (1) ◽  
pp. L43-L55 ◽  
Author(s):  
Anders Andersson ◽  
Apostolos Bossios ◽  
Carina Malmhäll ◽  
Margareta Sjöstrand ◽  
Maria Eldh ◽  
...  
Keyword(s):  
Free Radicals ◽  
Human Blood ◽  
Tobacco Smoke ◽  
Oxygen Free Radicals ◽  
De Novo ◽  
Water Soluble ◽  
Cellular Functions ◽  
Short Term Exposure ◽  
Human Airways

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8+ and CD4+ cells as well as the CD4+ chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8+ cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8+ cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8+ cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8+ cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8+ cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a β-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8+ cells. OFR may play a key role in this context.

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

2016 ◽  
Vol 35 (7) ◽  
pp. 1474-1490 ◽  
Author(s):  
Anna Antosiewicz ◽  
Adam Jarmuła ◽  
Dorota Przybylska ◽  
Grażyna Mosieniak ◽  
Joanna Szczepanowska ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dihydrofolate Reductase ◽  
Thymidylate Synthase ◽  
Human Dihydrofolate Reductase

scholarly journals Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis in Borrelia spp

2006 ◽  
Vol 188 (3) ◽  
pp. 909-918 ◽  
Author(s):  
Jianmin Zhong ◽  
Stephane Skouloubris ◽  
Qiyuan Dai ◽  
Hannu Myllykallio ◽  
Alan G. Barbour
Keyword(s):  
Thymidylate Synthase ◽  
De Novo ◽  
Linear Plasmid ◽  
Borrelia Hermsii ◽  
Borrelia Garinii ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis

ABSTRACT The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.

scholarly journals Hydroxylamine Assimilation byRhodobacter capsulatusE1F1

2004 ◽  
Vol 279 (44) ◽  
pp. 45485-45494 ◽  
Author(s):  
Purificación Cabello ◽  
Carmen Pino ◽  
M. Francisca Olmo-Mira ◽  
Francisco Castillo ◽  
M. Dolores Roldán ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Methyl Viologen ◽  
Rhodobacter Capsulatus ◽  
Genomic Library ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Resting Cells

Rhodobacter capsulatusE1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including thehcpgene coding for a hybrid cluster protein (HCP). Expression ofhcpis probably regulated by a nitrite-sensitive repressor encoded by the adjacentnsrRgene. A His6-HCP was overproduced inEscherichia coliand purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammoniain vitrowith methyl viologen as reductant. The apparentKmvalues for NH2OH and methyl viologen were 1 mmand 7 μm, respectively, at the pH and temperature optima (9.3 and 40 °C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents.R. capsulatusE1F1 grew phototrophically, but not heterotrophically, with 1 mmNH2OH as nitrogen source, and up to 10 mmNH2OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented byl-methionine-d,l-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However,R. capsulatusB10S, a strain lacking the wholehcp-nasregion, did not grow with 1 mmNH2OH. Also,E. colicells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH2OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.

scholarly journals Potent and Selective Activity of a Combination of Thymidine and 1843U89, a Folate-Based Thymidylate Synthase Inhibitor, against Plasmodium falciparum

2000 ◽  
Vol 44 (4) ◽  
pp. 1047-1050 ◽  
Author(s):  
Lei Jiang ◽  
Pei-Chieh Lee ◽  
John White ◽  
Pradipsinh K. Rathod
Keyword(s):  
Plasmodium Falciparum ◽  
Thymidylate Synthase ◽  
Mammalian Cells ◽  
De Novo ◽  
Antimalarial Activity ◽  
Malarial Parasite ◽  
Drug Resistant ◽  
Additional Tool ◽  
Synthase Inhibitor

ABSTRACT Unlike mammalian cells, malarial parasites are completely dependent on the de novo pyrimidine pathway and lack the enzymes to salvage preformed pyrimidines. In the present study, first, it is shown that 1843U89, even without polyglutamylation, is a potent folate-based inhibitor of purified malarial parasite thymidylate synthase. The binding was noncompetitive with respect to methylenetetrahydrofolate, and 1843U89 had a Ki of 1 nM. The compound also had potent antimalarial activity in vitro. Plasmodium falciparum cells in culture were inhibited by 1843U89, with a 50% inhibitory concentration of about 70 nM. The compound was effective against drug-sensitive as well as drug-resistant clones ofP. falciparum. As predicted by the biochemistry of the parasite, the potent inhibition of parasite proliferation by 1843U89 could not be reversed with 10 μM thymidine. In contrast, in the presence of 10 μM thymidine, mammalian cells were unaffected by 1843U89 even at concentrations as high as 0.1 mM, thus offering a selectivity window of more than 1,000-fold. On this basis, folate-based thymidylate synthase inhibitors may represent a powerful additional tool that can be used to combat drug-resistant malaria.

scholarly journals Abnormal deoxyuridine suppression test in congenital methylmalonic aciduria-homocystinuria without megaloblastic anemia: divergent biochemical and morphological bone marrow manifestations of disordered cobalamin metabolism in man

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 306-311 ◽  
Author(s):  
R Carmel ◽  
SI Goodman
Keyword(s):  
De Novo ◽  
Morphological Changes ◽  
Megaloblastic Anemia ◽  
Stress Test ◽  
Methylmalonic Aciduria ◽  
Cobalamin Deficiency ◽  
Suppression Test ◽  
Cobalamin Metabolism ◽  
Thymidylate Synthesis

Abstract We studied two brothers (J.R. and M.R.) with the cobalamin D variant of congenital methylmalonic aciduria-homocystinuria, whose previously reported lack of megaloblastic anemia conflicted with current concepts of cobalamin's role in DNA synthesis and the “methyltetrahydrofolate (MTHF) trap” hypothesis. Both subjects were indeed hematologically normal, although J.R. had a mean corpuscular volume of 96 fl. However, both demonstrated abnormalities in the deoxyuridine suppression test. J.R. had an abnormal suppression value of 21.0% (normal less than 10%) that was correctable by adding hydroxocobalamin or folic acid in vitro but not MTHF. M.R. had normal suppression (8.9%), but demonstrated worsening (18.6%) when MTHF was added. J.R.'s classical deoxyuridine suppression pattern of cobalamin deficiency thus supports the trap hypothesis. However, his lack of comparable morphological changes suggests that impaired de novo thymidylate synthesis and the trap hypothesis, though valid, may not fully account for the megaloblastic maturation accompanying cobalamin deficiency. Equally noteworthy was the deleterious effect of MTHF on M.R.'s marrow, suggesting its potential usefulness as an in vitro “stress test” for latent cobalamin abnormality.

Sign in / Sign up

Export Citation Format

Share Document