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Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

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CTP levels probed by mass spectrometry after addition of C13-labeled cytidine to the media.Unlabeled C12-CTP population represents the proportion of CTP synthesized by CtpS from cellular pools of UTP. The CtpSE277R has a higher intracellular C12-CTP pool both at the initial time point as well as at the end of the time course, where C12-CTP is almost twice as high as in the wild-type strain.DOI:http://dx.doi.org/10.7554/eLife.03638.031
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fig8s2: CTP levels probed by mass spectrometry after addition of C13-labeled cytidine to the media.Unlabeled C12-CTP population represents the proportion of CTP synthesized by CtpS from cellular pools of UTP. The CtpSE277R has a higher intracellular C12-CTP pool both at the initial time point as well as at the end of the time course, where C12-CTP is almost twice as high as in the wild-type strain.DOI:http://dx.doi.org/10.7554/eLife.03638.031

Mentions: As predicted based on the independence of nucleoside import from nucleotide biosynthesis, the incorporation of C13-label into the CTP pool was similar in the wild type and CtpSE277R strains, indicating that both take up labeled cytidine and convert it into CTP at approximately the same rate (Figure 8B). In wild type cells, as the C13-CTP pool increased, the fraction of C12-CTP sharply decreased (Figure 8C). Thus, feedback regulation mechanisms compensate for the increased CTP production from cytidine by reducing de novo CTP production by CtpS. The decrease in the fraction of unlabeled CTP was less pronounced in the CtpSE277R mutant and by the end of the period assayed, unlabeled CTP levels were almost twofold higher in the CtpSE377R strain than in wild type (Figure 8—figure supplement 2). This result supports our conclusion that CtpSE277R hyperactivates CtpS by disrupting its negative feedback regulation and that this hyperactivation more than compensates for its reduced enzymatic activity. Since disruption of just one interaction in the proposed polymerization interface weakened the ability of CtpS to control CTP production even when all other forms of CtpS regulation are unaltered, we predict that any disruption of regions of inter-tetrameric contact, either by changes to the protein sequence or by chemical perturbation, would cause this deleterious regulatory defect.


Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

CTP levels probed by mass spectrometry after addition of C13-labeled cytidine to the media.Unlabeled C12-CTP population represents the proportion of CTP synthesized by CtpS from cellular pools of UTP. The CtpSE277R has a higher intracellular C12-CTP pool both at the initial time point as well as at the end of the time course, where C12-CTP is almost twice as high as in the wild-type strain.DOI:http://dx.doi.org/10.7554/eLife.03638.031
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126345&req=5

fig8s2: CTP levels probed by mass spectrometry after addition of C13-labeled cytidine to the media.Unlabeled C12-CTP population represents the proportion of CTP synthesized by CtpS from cellular pools of UTP. The CtpSE277R has a higher intracellular C12-CTP pool both at the initial time point as well as at the end of the time course, where C12-CTP is almost twice as high as in the wild-type strain.DOI:http://dx.doi.org/10.7554/eLife.03638.031
Mentions: As predicted based on the independence of nucleoside import from nucleotide biosynthesis, the incorporation of C13-label into the CTP pool was similar in the wild type and CtpSE277R strains, indicating that both take up labeled cytidine and convert it into CTP at approximately the same rate (Figure 8B). In wild type cells, as the C13-CTP pool increased, the fraction of C12-CTP sharply decreased (Figure 8C). Thus, feedback regulation mechanisms compensate for the increased CTP production from cytidine by reducing de novo CTP production by CtpS. The decrease in the fraction of unlabeled CTP was less pronounced in the CtpSE277R mutant and by the end of the period assayed, unlabeled CTP levels were almost twofold higher in the CtpSE377R strain than in wild type (Figure 8—figure supplement 2). This result supports our conclusion that CtpSE277R hyperactivates CtpS by disrupting its negative feedback regulation and that this hyperactivation more than compensates for its reduced enzymatic activity. Since disruption of just one interaction in the proposed polymerization interface weakened the ability of CtpS to control CTP production even when all other forms of CtpS regulation are unaltered, we predict that any disruption of regions of inter-tetrameric contact, either by changes to the protein sequence or by chemical perturbation, would cause this deleterious regulatory defect.

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

Show MeSH