Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A domino effect in antifolate drug action in Escherichia coli

Abstract

Mass spectrometry technologies for measurement of cellular metabolism are opening new avenues to explore drug activity. Trimethoprim is an antibiotic that inhibits bacterial dihydrofolate reductase (DHFR). Kinetic flux profiling with 15N-labeled ammonia in Escherichia coli reveals that trimethoprim leads to blockade not only of DHFR but also of another critical enzyme of folate metabolism: folylpoly-γ-glutamate synthetase (FP-γ-GS). Inhibition of FP-γ-GS is not directly due to trimethoprim. Instead, it arises from accumulation of DHFR's substrate dihydrofolate, which we show is a potent FP-γ-GS inhibitor. Thus, owing to the inherent connectivity of the metabolic network, falling DHFR activity leads to falling FP-γ-GS activity in a domino-like cascade. This cascade results in complex folate dynamics, and its incorporation in a computational model of folate metabolism recapitulates the dynamics observed experimentally. These results highlight the potential for quantitative analysis of cellular metabolism to reveal mechanisms of drug action.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Changes in folate pools with the addition of trimethoprim (4 μg ml−1).
Figure 2: Probing folate fluxes in the presence of trimethoprim.
Figure 3: DHF inhibits FP-γ-GS activity, as determined by in vitro FP-γ-GS assays.
Figure 4: Computational modeling of cellular folate dynamics.

References

  1. Voet, D. & Voet, J.G. in Biochemistry (eds. Harris, D. & Fitzgerald, P.) 482–486 (John Wiley & Sons, Inc., Hoboken, New Jersey, USA, 2004).

    Google Scholar 

  2. Quinlivan, E.P., McPartlin, J., Weir, D.G. & Scott, J. Mechanism of the antimicrobial drug trimethoprim revisited. FASEB J. 14, 2519–2524 (2000).

    CAS  Article  Google Scholar 

  3. Sabatine, M.S. et al. Metabolomic identification of novel biomarkers of myocardial ischemia. Circulation 112, 3868–3875 (2005).

    CAS  Article  Google Scholar 

  4. Bajad, S.U. et al. Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J. Chromatogr. A. 1125, 76–88 (2006).

    CAS  Article  Google Scholar 

  5. van der Werf, M.J., Overkamp, K.M., Muilwijk, B., Coulier, L. & Hankemeier, T. Microbial metabolomics: toward a platform with full metabolome coverage. Anal. Biochem. 370, 17–25 (2007).

    CAS  Article  Google Scholar 

  6. Sauer, U. Metabolic networks in motion: 13C-based flux analysis. Mol. Syst. Biol. 2, 62 (2006).

    Article  Google Scholar 

  7. Yuan, J., Fowler, W.U., Kimball, E., Lu, W. & Rabinowitz, J.D. Kinetic flux profiling of nitrogen assimilation in Escherichia coli. Nat. Chem. Biol. 2, 529–530 (2006).

    CAS  Article  Google Scholar 

  8. Noh, K. et al. Metabolic flux analysis at ultra short time scale: isotopically non-stationary 13C labeling experiments. J. Biotechnol. 129, 249–267 (2007).

    Article  Google Scholar 

  9. Bushby, S.R. & Hitchings, G.H. Trimethoprim, a sulphonamide potentiator. Br. J. Pharmacol. Chemother. 33, 72–90 (1968).

    CAS  Article  Google Scholar 

  10. Reeves, D.S. Sulphamethoxazole-trimethoprim: the first two years. J. Clin. Pathol. 24, 430–437 (1971).

    CAS  Article  Google Scholar 

  11. Then, R.L. History and future of antimicrobial diaminopyrimidines. J. Chemother. 5, 361–368 (1993).

    CAS  PubMed  Google Scholar 

  12. Gangjee, A., Jain, H.D. & Kurup, S. Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents: part I. Anticancer Agents Med. Chem. 7, 524–542 (2007).

    CAS  Article  Google Scholar 

  13. Schnell, J.R., Dyson, H.J. & Wright, P.E. Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu. Rev. Biophys. Biomol. Struct. 33, 119–140 (2004).

    CAS  Article  Google Scholar 

  14. Friedkin, M. Thymidylate synthetase. Adv. Enzymol. 38, 235–292 (1973).

    CAS  PubMed  Google Scholar 

  15. Pogolotti, A.L. & Santi, D.V. in Bioorganic Chemistry Vol. 1 (ed. van Tamelen, E.E.) 277–311 (Academic Press, New York, 1977).

    Google Scholar 

  16. Danenberg, P.V. Thymidylate synthetase - a target enzyme in cancer chemotherapy. Biochim. Biophys. Acta 473, 73–92 (1977).

    CAS  PubMed  Google Scholar 

  17. Santi, D.V. & Danenberg, P.V. in Folates and Pterins Vol. 1 (eds. Blackley, R.L. & Benkovic, S.J.) 345–398 (John Wiley & Sons, New York, 1984).

    Google Scholar 

  18. Carreras, C.W. & Santi, D.V. The catalytic mechanism and structure of thymidylate synthase. Annu. Rev. Biochem. 64, 721–762 (1995).

    CAS  Article  Google Scholar 

  19. McGuire, J.J. & Bertino, J.R. Enzymatic synthesis and function of folylpolyglutamates. Mol. Cell. Biochem. 38, 19–48 (1981).

    CAS  Article  Google Scholar 

  20. McGuire, J.J. & Coward, J.K. in Folates and Pterins Vol. 1 (eds. Blackley, R.L. & Benkovic, S.J.) 135–190 (John Wiley & Sons, New York, 1984).

    Google Scholar 

  21. Shane, B. Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism. Vitam. Horm. 45, 263–335 (1989).

    CAS  Article  Google Scholar 

  22. Lowe, K.E. et al. Regulation of folate and one-carbon metabolism in mammalian cells. II. Effect of folylpoly-gamma-glutamate synthetase substrate specificity and level on folate metabolism and folylpoly-gamma-glutamate specificity of metabolic cycles of one-carbon metabolism. J. Biol. Chem. 268, 21665–21673 (1993).

    CAS  PubMed  Google Scholar 

  23. Quinlivan, E.P., Hanson, A.D. & Gregory, J.F. The analysis of folate and its metabolic precursors in biological samples. Anal. Biochem. 348, 163–184 (2006).

    CAS  Article  Google Scholar 

  24. Ferone, R., Hanlon, M.H., Singer, S.C. & Hunt, D.F. α-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli. J. Biol. Chem. 261, 16356–16362 (1986).

    CAS  PubMed  Google Scholar 

  25. Ferone, R., Singer, S.C. & Hunt, D.F. In vitro synthesis of α-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli. J. Biol. Chem. 261, 16363–16371 (1986).

    CAS  PubMed  Google Scholar 

  26. Bognar, A.L., Osborne, C., Shane, B., Singer, S.C. & Ferone, R. Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. J. Biol. Chem. 260, 5625–5630 (1985).

    CAS  PubMed  Google Scholar 

  27. Garratt, L.C. et al. Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry. Rapid Commun. Mass Spectrom. 19, 2390–2398 (2005).

    CAS  Article  Google Scholar 

  28. Lu, W., Kwon, Y.K. & Rabinowitz, J.D. Isotope ratio-based profiling of microbial folates. J. Am. Soc. Mass Spectrom. 18, 898–909 (2007).

    CAS  Article  Google Scholar 

  29. Scott, J.M. in Folates and Pterins Vol. 1 (eds. Blackley, R.L. & Benkovic, S.J.) 307–327 (John Wiley & Sons, New York, 1984).

    Google Scholar 

  30. Suh, J.R., Oppenheim, E.W., Girgis, S. & Stover, P.J. Purification and properties of a folate-catabolizing enzyme. J. Biol. Chem. 275, 35646–35655 (2000).

    CAS  Article  Google Scholar 

  31. Brauer, M.J. et al. Conservation of the metabolomic response to starvation across two divergent microbes. Proc. Natl. Acad. Sci. USA 103, 19302–19307 (2006).

    CAS  Article  Google Scholar 

  32. Kisliuk, R.L. Pteroylpolyglutamates. Mol. Cell. Biochem. 39, 331–345 (1981).

    CAS  Article  Google Scholar 

  33. Hsieh, Y. HPLC-MS/MS in drug metabolism and pharmacokinetic screening. Expert Opin. Drug Metab. Toxicol. 4, 93–101 (2008).

    CAS  Article  Google Scholar 

  34. Clayton, T.A. et al. Pharmaco-metabonomic phenotyping and personalized drug treatment. Nature 440, 1073–1077 (2006).

    CAS  Article  Google Scholar 

  35. Masurekar, M. & Brown, G.M. Partial purification and properties of an enzyme from Escherichia coli that catalyzes the conversion of glutamic acid and 10-formyltetrahydropteroylglutamic acid to 10-formyltetrahydropteroyl-gamma-glutamylglutamic acid. Biochemistry 14, 2424–2430 (1975).

    CAS  Article  Google Scholar 

  36. Allegra, C.J., Drake, J.C., Jolivet, J. & Chabner, B.A. Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates. Proc. Natl. Acad. Sci. USA 82, 4881–4885 (1985).

    CAS  Article  Google Scholar 

  37. Allegra, C.J., Hoang, K., Yeh, G.C., Drake, J.C. & Baram, J. Evidence for direct inhibition of de novo purine synthesis in human MCF- 7 breast cells as a principal mode of metabolic inhibition by methotrexate. J. Biol. Chem. 262, 13520–13526 (1987).

    CAS  PubMed  Google Scholar 

  38. Matthews, R.G. & Baugh, C.M. Interactions of pig liver methylenetetrahydrofolate reductase with methylenetetrahydropteroylpolyglutamate substrates and with dihydropteroylpolyglutamate inhibitors. Biochemistry 19, 2040–2045 (1980).

    CAS  Article  Google Scholar 

  39. Kisliuk, R.L., Gaumont, Y. & Baugh, C.M. Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase. J. Biol. Chem. 249, 4100–4103 (1974).

    CAS  PubMed  Google Scholar 

  40. Dolnick, B.J. & Cheng, Y.C. Human thymidylate synthetase. II. Derivatives of pteroylmono- and -polyglutamates as substrates and inhibitors. J. Biol. Chem. 253, 3563–3567 (1978).

    CAS  PubMed  Google Scholar 

  41. Matthews, R.G. & Haywood, B.J. Inhibition of pig liver methylenetetrahydrofolate reductase by dihydrofolate: some mechanistic and regulatory implications. Biochemistry 18, 4845–4851 (1979).

    CAS  Article  Google Scholar 

  42. Reed, M.C., Nijhout, H.F., Sparks, R. & Ulrich, C.M. A mathematical model of the methionine cycle. J. Theor. Biol. 226, 33–43 (2004).

    CAS  Article  Google Scholar 

  43. Reed, M.C. et al. A mathematical model gives insights into nutritional and genetic aspects of folate-mediated one-carbon metabolism. J. Nutr. 136, 2653–2661 (2006).

    CAS  Article  Google Scholar 

  44. 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 

  45. Ibarra, R.U., Edwards, J.S. & Palsson, B.O. Escherichia coli K-12 undergoes adaptive evolution to achieve in silico predicted optimal growth. Nature 420, 186–189 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research was supported by the US National Institutes of Health (NIH) Center for Quantitative Biology at Princeton University (P50GM071508). Additional support came from the Beckman Foundation, the US National Science Foundation (NSF) Dynamic Data Driven Applications Systems grant CNS-0540181, the American Heart Association grant 0635188N, NSF Career Award MCB-0643859 and the NIH grant AI078063 (to J.D.R.).

Author information

Authors and Affiliations

Authors

Contributions

Y.K.K. and J.D.R. designed experiments, analyzed data and wrote the paper. W.L. developed the LC-MS/MS method. E.M. wrote the computer code. N.K. and A.B. contributed to biochemical assays of FP-γ-GS activity. A.B. edited the paper.

Corresponding author

Correspondence to Joshua D Rabinowitz.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods (PDF 363 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kwon, Y., Lu, W., Melamud, E. et al. A domino effect in antifolate drug action in Escherichia coli. Nat Chem Biol 4, 602–608 (2008). https://doi.org/10.1038/nchembio.108

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.108

Further reading

Search

Quick links

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