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Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

Pisithkul T, Jacobson TB, O'Brien TJ, Stevenson DM, Amador-Noguez D - Appl. Environ. Microbiol. (2015)

Bottom Line: Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels.We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways.Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA Great Lake Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.

No MeSH data available.


Related in: MedlinePlus

Carbon flux into purine and pyrimidine biosynthetic pathways is inhibited by phenolic amides. (A) Using LC-MS, we followed the dynamic incorporation of 13C from [1,2-13C]xylose into downstream metabolites after exposure to phenolic amides or ferulic acid. Exponential-phase E. coli cultures growing anaerobically on nonlabeled xylose were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching them from nonlabeled xylose to [1,2-13C]xylose. Samples for intracellular metabolite analysis were then taken at 0.5, 1, 2, 4, 7, 10, and 15 min thereafter. The blue-to-red gradient represents the amount of 13C label incorporation (sum of all 13C-labeled forms) into each metabolite as a fraction of its total pool of the control sample; for each metabolite, the lowest value (nonlabeled) was set to blue, while the highest 13C-label fraction value in control samples was set to red. The data represent the average of two biological replicates. (B) E. coli cells were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching to [1,2-13C]xylose. The x axis represents minutes after the switch to [1,2-13C]xylose, and the y axis represents the fraction of the observed compound of the indicated isotopic form (containing different number of 13C atoms). The data shown are representative of two biological replicates.
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Figure 2: Carbon flux into purine and pyrimidine biosynthetic pathways is inhibited by phenolic amides. (A) Using LC-MS, we followed the dynamic incorporation of 13C from [1,2-13C]xylose into downstream metabolites after exposure to phenolic amides or ferulic acid. Exponential-phase E. coli cultures growing anaerobically on nonlabeled xylose were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching them from nonlabeled xylose to [1,2-13C]xylose. Samples for intracellular metabolite analysis were then taken at 0.5, 1, 2, 4, 7, 10, and 15 min thereafter. The blue-to-red gradient represents the amount of 13C label incorporation (sum of all 13C-labeled forms) into each metabolite as a fraction of its total pool of the control sample; for each metabolite, the lowest value (nonlabeled) was set to blue, while the highest 13C-label fraction value in control samples was set to red. The data represent the average of two biological replicates. (B) E. coli cells were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching to [1,2-13C]xylose. The x axis represents minutes after the switch to [1,2-13C]xylose, and the y axis represents the fraction of the observed compound of the indicated isotopic form (containing different number of 13C atoms). The data shown are representative of two biological replicates.

Mentions: As shown in Fig. 2, the incorporation of 13C into metabolites in the PPP, glycolysis, or reductive part of the TCA cycle was not greatly affected in cells exposed to feruloyl or coumaroyl amide. Specifically, the 13C-labeling dynamics of the PPP intermediates xylulose-5-phosphate, ribose-5-phosphate, and d-sedoheptulose-7-phosphate were comparable between controls and feruloyl or coumaroyl amide-treated samples. This pattern was also true for all measured glycolytic intermediates, including glucose-6-phosphate (G6P), fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), and the downstream metabolite acetyl coenzyme A (acetyl-CoA). Similarly, 13C-labeling dynamics of metabolites in the reductive TCA cycle (i.e., malate, fumarate, and succinate) were only minimally affected.


Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

Pisithkul T, Jacobson TB, O'Brien TJ, Stevenson DM, Amador-Noguez D - Appl. Environ. Microbiol. (2015)

Carbon flux into purine and pyrimidine biosynthetic pathways is inhibited by phenolic amides. (A) Using LC-MS, we followed the dynamic incorporation of 13C from [1,2-13C]xylose into downstream metabolites after exposure to phenolic amides or ferulic acid. Exponential-phase E. coli cultures growing anaerobically on nonlabeled xylose were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching them from nonlabeled xylose to [1,2-13C]xylose. Samples for intracellular metabolite analysis were then taken at 0.5, 1, 2, 4, 7, 10, and 15 min thereafter. The blue-to-red gradient represents the amount of 13C label incorporation (sum of all 13C-labeled forms) into each metabolite as a fraction of its total pool of the control sample; for each metabolite, the lowest value (nonlabeled) was set to blue, while the highest 13C-label fraction value in control samples was set to red. The data represent the average of two biological replicates. (B) E. coli cells were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching to [1,2-13C]xylose. The x axis represents minutes after the switch to [1,2-13C]xylose, and the y axis represents the fraction of the observed compound of the indicated isotopic form (containing different number of 13C atoms). The data shown are representative of two biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4551265&req=5

Figure 2: Carbon flux into purine and pyrimidine biosynthetic pathways is inhibited by phenolic amides. (A) Using LC-MS, we followed the dynamic incorporation of 13C from [1,2-13C]xylose into downstream metabolites after exposure to phenolic amides or ferulic acid. Exponential-phase E. coli cultures growing anaerobically on nonlabeled xylose were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching them from nonlabeled xylose to [1,2-13C]xylose. Samples for intracellular metabolite analysis were then taken at 0.5, 1, 2, 4, 7, 10, and 15 min thereafter. The blue-to-red gradient represents the amount of 13C label incorporation (sum of all 13C-labeled forms) into each metabolite as a fraction of its total pool of the control sample; for each metabolite, the lowest value (nonlabeled) was set to blue, while the highest 13C-label fraction value in control samples was set to red. The data represent the average of two biological replicates. (B) E. coli cells were pretreated with 5.5 mM feruloyl amide, coumaroyl amide, or ferulic acid for 5 min before switching to [1,2-13C]xylose. The x axis represents minutes after the switch to [1,2-13C]xylose, and the y axis represents the fraction of the observed compound of the indicated isotopic form (containing different number of 13C atoms). The data shown are representative of two biological replicates.
Mentions: As shown in Fig. 2, the incorporation of 13C into metabolites in the PPP, glycolysis, or reductive part of the TCA cycle was not greatly affected in cells exposed to feruloyl or coumaroyl amide. Specifically, the 13C-labeling dynamics of the PPP intermediates xylulose-5-phosphate, ribose-5-phosphate, and d-sedoheptulose-7-phosphate were comparable between controls and feruloyl or coumaroyl amide-treated samples. This pattern was also true for all measured glycolytic intermediates, including glucose-6-phosphate (G6P), fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), and the downstream metabolite acetyl coenzyme A (acetyl-CoA). Similarly, 13C-labeling dynamics of metabolites in the reductive TCA cycle (i.e., malate, fumarate, and succinate) were only minimally affected.

Bottom Line: Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels.We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways.Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA Great Lake Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.

No MeSH data available.


Related in: MedlinePlus