Pyrimidine homeostasis is accomplished by directed overflow metabolism

Abstract

Cellular metabolism converts available nutrients into usable energy and biomass precursors. The process is regulated to facilitate efficient nutrient use and metabolic homeostasis. Feedback inhibition of the first committed step of a pathway by its final product is a classical means of controlling biosynthesis1,2,3,4. In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate1,4,5. The physiological consequences of disrupting this regulation, however, have not been previously explored. Here we identify an alternative regulatory strategy that enables precise control of pyrimidine pathway end-product levels, even in the presence of dysregulated biosynthetic flux. The mechanism involves cooperative feedback regulation of the near-terminal pathway enzyme uridine monophosphate kinase6. Such feedback leads to build-up of the pathway intermediate uridine monophosphate, which is in turn degraded by a conserved phosphatase, here termed UmpH, with previously unknown physiological function7,8. Such directed overflow metabolism allows homeostasis of uridine triphosphate and cytidine triphosphate levels at the expense of uracil excretion and slower growth during energy limitation. Disruption of the directed overflow regulatory mechanism impairs growth in pyrimidine-rich environments. Thus, pyrimidine homeostasis involves dual regulatory strategies, with classical feedback inhibition enhancing metabolic efficiency and directed overflow metabolism ensuring end-product homeostasis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Increased pyrimidine flux triggers overflow to uracil.
Figure 2: Pyrimidine overflow pathway is initiated by catabolism of UMP by UmpH.
Figure 3: Cooperative inhibition of UMP kinase by UTP maintains end-product homeostasis.
Figure 4: Directed overflow metabolism in biosynthesis is analogous to central carbon overflow metabolism.

References

  1. 1

    Gerhart, J. C. & Pardee, A. B. The enzymology of control by feedback inhibition. J. Biol. Chem. 237, 891–896 (1962)

    CAS  PubMed  Google Scholar 

  2. 2

    Savageau, M. A. Optimal design of feedback-control by inhibition — dynamic considerations. J. Mol. Evol. 5, 199–222 (1975)

    ADS  CAS  PubMed  Google Scholar 

  3. 3

    Umbarger, H. E. Evidence for a negative-feedback mechanism in the biosynthesis of isoleucine. Science 123, 848–849 (1956)

    ADS  CAS  PubMed  Google Scholar 

  4. 4

    Pardee, A. B. & Yates, R. A. Control of pyrimidine biosynthesis in Escherichia coli by a feed-back mechanism. J. Biol. Chem. 221, 757–770 (1956)

    CAS  PubMed  Google Scholar 

  5. 5

    Kantrowitz, E. R. Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase. Arch. Biochem. Biophys. 519, 81–90 (2012)

    CAS  PubMed  Google Scholar 

  6. 6

    Meyer, P. et al. Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP. J. Biol. Chem. 283, 36011–36018 (2008)

    CAS  PubMed  Google Scholar 

  7. 7

    Kuznetsova, E. et al. Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family. J. Biol. Chem. 281, 36149–36161 (2006)

    CAS  PubMed  Google Scholar 

  8. 8

    Proudfoot, M. et al. General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG. J. Biol. Chem. 279, 54687–54694 (2004)

    CAS  PubMed  Google Scholar 

  9. 9

    Orth, J. D. et al. A comprehensive genome-scale reconstruction of Escherichia coli metabolism—2011. Mol. Syst. Biol. 7, 535 (2011)

    PubMed  PubMed Central  Google Scholar 

  10. 10

    Fell, D. Understanding the Control of Metabolism (Portland Press, 1997)

    Google Scholar 

  11. 11

    Kacser, H., Burns, J. A. & Fell, D. A. The control of flux. Biochem. Soc. Trans. 23, 341–366 (1995)

    CAS  PubMed  Google Scholar 

  12. 12

    Heinrich, R. & Rapoport, T. A. Linear steady-state treatment of enzymatic chains. General properties, control and effector strength. Eur. J. Biochem. 42, 89–95 (1974)

    CAS  PubMed  Google Scholar 

  13. 13

    Crabtree, B. & Newsholme, E. A. The derivation and interpretation of control coefficients. Biochem. J. 247, 113–120 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Small, J. R. & Kacser, H. Responses of metabolic systems to large changes in enzyme-activities and effectors. 1. The linear treatment of unbranched chains. Eur. J. Biochem. 213, 613–624 (1993)

    CAS  PubMed  Google Scholar 

  15. 15

    Kell, D. B. & Westerhoff, H. V. Metabolic control theory: its role in microbiology and biotechnology. FEMS Microbiol. Rev. 39, 305–320 (1986)

    CAS  Google Scholar 

  16. 16

    Hofmeyr, J. H. S. & Cornish-Bowden, A. Quantitative assessment of regulation in metabolic systems. Eur. J. Biochem. 200, 223–236 (1991)

    CAS  PubMed  Google Scholar 

  17. 17

    Keseler, I. M. et al. EcoCyc: a comprehensive database of Escherichia coli biology. Nucleic Acids Res. 39, D583–D590 (2011)

    CAS  PubMed  Google Scholar 

  18. 18

    Peterson, A. W., Cockrell, G. M. & Kantrowitz, E. R. A second allosteric site in Escherichia coli aspartate transcarbamoylase. Biochemistry 51, 4776–4778 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Wild, J. R., Loughrey-Chen, S. J. & Corder, T. S. In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase. Proc. Natl Acad. Sci. USA 86, 46–50 (1989)

    ADS  CAS  PubMed  Google Scholar 

  20. 20

    Anderson, P. M. & Meister, A. Control of Escherichia coli carbamyl phosphate synthetase by purine and pyrimidine nucleotides. Biochemistry 5, 3164–3169 (1966)

    CAS  PubMed  Google Scholar 

  21. 21

    Delannay, S. et al. Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways. J. Mol. Biol. 286, 1217–1228 (1999)

    CAS  PubMed  Google Scholar 

  22. 22

    Loh, K. D. et al. A previously undescribed pathway for pyrimidine catabolism. Proc. Natl Acad. Sci. USA 103, 5114–5119 (2006)

    ADS  CAS  PubMed  Google Scholar 

  23. 23

    Bennett, B. D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nature Chem. Biol. 5, 593–599 (2009)

    CAS  Google Scholar 

  24. 24

    Flamholz, A., Noor, E., Bar-Even, A. & Milo, R. eQuilibrator–the biochemical thermodynamics calculator. Nucleic Acids Res. 40, D770–D775 (2012)

    CAS  PubMed  Google Scholar 

  25. 25

    Bianchi, V. & Spychala, J. Mammalian 5′-nucleotidases. J. Biol. Chem. 278, 46195–46198 (2003)

    CAS  PubMed  Google Scholar 

  26. 26

    Tremblay, L. W., Dunaway-Mariano, D. & Allen, K. N. Structure and activity analyses of Escherichia coli K-12 NagD provide insight into the evolution of biochemical function in the haloalkanoic acid dehalogenase superfamily. Biochemistry 45, 1183–1193 (2006)

    CAS  PubMed  Google Scholar 

  27. 27

    Bucurenci, N. et al. Mutational analysis of UMP kinase from Escherichia coli. J. Bacteriol. 180, 473–477 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Sauer, U. & Eikmanns, B. J. The PEP–pyruvate–oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rev. 29, 765–794 (2005)

    CAS  PubMed  Google Scholar 

  29. 29

    Roche, T. E. & Hiromasa, Y. Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer. Cell. Mol. Life Sci. 64, 830–849 (2007)

    CAS  PubMed  Google Scholar 

  30. 30

    Soupene, E. et al. Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression. J. Bacteriol. 185, 5611–5626 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Silhavy, T. J., Berman, M. L. & Enquist, L. W. Experiments With Gene Fusions (Cold Spring Harbor Press, 1984)

    Google Scholar 

  32. 32

    Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006)

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

    ADS  CAS  PubMed  Google Scholar 

  34. 34

    Kitagawa, M. et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete Set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 12, 291–299 (2006)

    Google Scholar 

  35. 35

    Gutnick, D., Calvo, J. M., Klopotowski, T. & Ames, B. N. Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT-2. J. Bacteriol. 100, 215–219 (1969)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Neidhardt, F. C., Bloch, P. L. & Smith, D. F. Culture medium for enterobacteria. J. Bacteriol. 119, 736–747 (1974)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Rabinowitz, J. D. & Kimball, E. Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal. Chem. 79, 6167–6173 (2007)

    CAS  PubMed  Google Scholar 

  38. 38

    Bennett, B. D., Yuan, J., Kimball, E. H. & Rabinowitz, J. D. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nature Protocols 3, 1299–1311 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Lu, W. et al. Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer. Anal. Chem. 82, 3212–3221 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Lu, W., Kimball, E. & Rabinowitz, J. D. A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J. Am. Soc. Mass Spectrom. 17, 37–50 (2006)

    CAS  PubMed  Google Scholar 

  41. 41

    Clasquin, M. F., Melamud, E. & Rabinowitz, J. D. LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. Curr. Protoc. Bioinformatics 14, 14.11 (2012)

    Google Scholar 

  42. 42

    Goodarzi, H. et al. Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli. Mol. Syst. Biol. 6, 378 (2010)

    PubMed  PubMed Central  Google Scholar 

  43. 43

    Goodarzi, H., Elemento, O. & Tavazoie, S. Revealing global regulatory perturbations across human cancers. Mol. Cell 36, 900–911 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

M.L.R. was supported by an National Science foundation (NSF) Graduate Research Fellowship. J.D.R. was supported by NSF CDI Award CBET-0941143 and CAREER Award MCB-0643859, and DOE-AFOSR Award DE-SC0002077/FA9550-09-1-0580, and an American Heart Association Scientist Development Grant.

Author information

Affiliations

Authors

Contributions

M.L.R., A.M.H., B.D.Y., Y.-F.X. and J.D.R. designed experiments and analyses. B.D.Y. generated feedback-dysregulated strains and performed experiments on regulation of the de novo pathway. A.M.H. performed competitions, microarrays and metabolite quantification. M.L.R. generated overflow and cooperativity mutants and measured their metabolites and growth. M.L.R. and J.D.R. wrote the paper with input from all authors.

Corresponding author

Correspondence to Joshua D. Rabinowitz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-6. (PDF 2376 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Reaves, M., Young, B., Hosios, A. et al. Pyrimidine homeostasis is accomplished by directed overflow metabolism. Nature 500, 237–241 (2013). https://doi.org/10.1038/nature12445

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing