Synthesis of FUDP-<i>N</i>-acetylglucosamine and FUDP-glucose in<i>Saccharomyces cerevisiae</i>cells treated with 5-fluorouracil

Sillero, Marı́a A. Günther; Pérez-Zúñiga, Francisco J.; Gomes, Joana; Carvalho, Ana Isabel De; Martins, Susana; Silles, Eduardo; Sillero, Antonio

doi:10.1111/j.1567-1364.2007.00321.x

Cited by 6 publications

(2 citation statements)

References 27 publications

(46 reference statements)

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“…Quantitative production of UNAG-3P, degradation of ATP and accumulation of ADP was determined by Maldi-TOF analysis as described [32]. ATP and UNAG were incubated or not with ζ toxin in buffer B (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl 2 ) for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Tabone¹,

Ayora²,

Alonso³

2014

Toxins

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Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.

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Section: Methodsmentioning

confidence: 99%

Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Tabone¹,

Ayora²,

Alonso³

2014

Toxins

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“…The addition of H 2 O 2 (0.05–1.0 mM) to the yeast cultures with low (0.025%) and high (2%) glucose concentration resulted in a sharp decrease in ATP and a less extensive decrease in GTP, CTP, UTP and ADP levels, respectively, [18,19] in fermentative conditions. According to our best knowledge, this effect in the context of 5-FU metabolism was studied only by Sillero and co-workers [20], who looked at levels of 5-fluoro-UDP-sugars in fermentative and oxidative (high and low concentrations of glucose, respectively) conditions.…”

Section: Introductionmentioning

confidence: 99%

Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions

et al. 2019

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The cytotoxic effect of 5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by fluorodeoxyuridine monophosphate (FdUMP). However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease in concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called ‘thymine-less death’ route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19F and 31P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours, however, the initial steady-state was preserved only for an hour, until the ATP concentration dropped by a half, which was monitored independently via 31P NMR spectra. After that, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast.

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Harvey

2011

Mass Spectrometry Reviews

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This review is the fifth update of the original review, published in 1999, on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2008. The first section of the review covers fundamental studies, fragmentation of carbohydrate ions, use of derivatives and new software developments for analysis of carbohydrate spectra. Among newer areas of method development are glycan arrays, MALDI imaging and the use of ion mobility spectrometry. The second section of the review discusses applications of MALDI MS to the analysis of different types of carbohydrate. Specific compound classes that are covered include carbohydrate polymers from plants, N- and O-linked glycans from glycoproteins, biopharmaceuticals, glycated proteins, glycolipids, glycosides and various other natural products. There is a short section on the use of MALDI mass spectrometry for the study of enzymes involved in glycan processing and a section on the use of MALDI MS to monitor products of the chemical synthesis of carbohydrates with emphasis on carbohydrate-protein complexes and glycodendrimers. Corresponding analyses by electrospray ionization now appear to outnumber those performed by MALDI and the amount of literature makes a comprehensive review on this technique impractical. However, most of the work relating to sample preparation and glycan synthesis is equally relevant to electrospray and, consequently, those proposing analyses by electrospray should also find material in this review of interest.

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Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose inSaccharomyces cerevisiaecells treated with 5-fluorouracil

Cited by 6 publications

References 27 publications

Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

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