Uptake of AMP, ADP, and ATP in<i>Escherichia coli</i>W

Watanabe, K.; Tomioka, Satsuki; Tanimura, Kiyoko; Oku, Hisae; Isoi, Koichiro

doi:10.1271/bbb.100063

Cited by 23 publications

(20 citation statements)

References 26 publications

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“…If there is a decrease of ATP concentration caused by an uptake by the bacteria, it is in a very small order of magnitude. A recent report supports the notion that an uptake of ATP by E. coli even with minimum medium is very slow [ 48 ]. Third, due to the complex nature of a whole-cell biocatalyst, the concentration of active protein cannot be determined exactly.…”

Section: Discussionsupporting

confidence: 57%

Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery

et al. 2015

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BackgroundHuman protein kinase CK2 represents a novel therapeutic target for neoplastic diseases. Inhibitors are in need to explore the druggability and the therapeutic options of this enzyme. A bottleneck in the search for new inhibitors is the availability of the target for testing. Therefore an assay was developed to provide easy access to CK2 for discovery of novel inhibitors.ResultsAutodisplay was used to present human CK2 on the surface of Escherichia coli. Heterotetrameric CK2 consists of two subunits, α and β, which were displayed individually on the surface. Co-display of CK2α and CK2β on the cell surface led to the formation of functional holoenzyme, as demonstrated by NaCl dependency of enzymatic activity, which differs from that of the catalytic subunit CK2α without β. In addition interaction of CK2α and CK2β at the cell surface was confirmed by co-immunoprecipitation assays. Surface displayed CK2 holoenzyme enabled an easy IC50 value determination. The IC50 values for the known CK2 inhibitors TBB and Silmitasertib were determined to be 50 and 3.3 nM, respectively.ConclusionSurface-displayed CK2α and CK2β assembled on the cell surface of E. coli to an active tetrameric holoenzyme. The whole-cell CK2 autodisplay assay as developed is suitable for inhibition studies. Furthermore, it can be used to determine quantitative CK2 inhibition data such as IC50 values. In summary, this is the first report on the functional surface display of a heterotetrameric enzyme on E. coli.

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Section: Discussionsupporting

confidence: 57%

Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery

et al. 2015

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“…However, when radiolabeled uridine was supplied to E. coli cells and cellular extracts were subsequently run on thin layer chromatography, a fast cleavage to the corresponding nucleobases was observed (see Supplemental Figure 1 online). Cleavage is assumed to occur in the periplasmic space of the bacterial cells by corresponding enzyme activities as shown for adenosine (Watanabe et al, 2011). Therefore, the question whether nucleosides are also substrates of PLUTO cannot be addressed with this experimental system.…”

Section: Biochemical Characterization Of Pluto a Nucleobase Transpormentioning

confidence: 99%

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

et al. 2012

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Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 mM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

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“…Nucleosides, which lack a phosphate group, are readily transported into the cytoplasm of V. cholerae via Nup transporters [Gumpenberger et al (accom-panying manuscript from separate group)]. However, no nucleotide transporter has been identified, suggesting that the phosphorylated nucleosides remain in the periplasm (Watanabe et al, 2011). Therefore, we hypothesized that expression of a periplasmic or extracellular phosphatase is required for growth on eDNA by releasing phosphate from the mononucleotides liberated by Xds and Dns.…”

Section: Introductionmentioning

confidence: 99%

Vibrio cholerae phosphatases required for the utilization of nucleotides and extracellular DNA as phosphate sources

McDonough

Kamp

Camilli

2015

Molecular Microbiology

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SummaryPhosphate is essential for life, being used in many core processes such as signal transduction and synthesis of nucleic acids. The waterborne agent of cholera, V ibrio cholerae, encounters phosphate limitation in both the aquatic environment and human intestinal tract. This bacterium can utilize extracellular DNA (eDNA) as a phosphate source, a phenotype dependent on secreted endo‐ and exonucleases. However, no transporter of nucleotides has been identified in V . cholerae, suggesting that in order for the organism to utilize the DNA as a phosphate source, it must first separate the phosphate and nucleoside groups before transporting phosphate into the cell. In this study, we investigated the factors required for assimilation of phosphate from eDNA. We identified PhoX, and the previously unknown proteins UshA and CpdB as the major phosphatases that allow phosphate acquisition from eDNA and nucleotides. We demonstrated separable but partially overlapping roles for the three phosphatases and showed that the activity of PhoX and CpdB is induced by phosphate limitation. Thus, this study provides mechanistic insight into how V . cholerae can acquire phosphate from extracellular DNA, which is likely to be an important phosphate source in the environment and during infection.

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Uptake of AMP, ADP, and ATP inEscherichia coliW

Cited by 23 publications

References 26 publications

Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery

Functional display of heterotetrameric human protein kinase CK2 on Escherichia coli: a novel tool for drug discovery

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

Vibrio cholerae phosphatases required for the utilization of nucleotides and extracellular DNA as phosphate sources

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