Skip to main content

Thank you for visiting 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 plausible metal-free ancestral analogue of the Krebs cycle composed entirely of α-ketoacids


Efforts to decipher the prebiotic roots of metabolic pathways have focused on recapitulating modern biological transformations, with metals typically serving in place of cofactors and enzymes. Here we show that the reaction of glyoxylate with pyruvate under mild aqueous conditions produces a series of α-ketoacid analogues of the reductive citric acid cycle without the need for metals or enzyme catalysts. The transformations proceed in the same sequence as the reverse Krebs cycle, resembling a protometabolic pathway, with glyoxylate acting as both the carbon source and reducing agent. Furthermore, the α-ketoacid analogues provide a natural route for the synthesis of amino acids by transamination with glycine, paralleling the extant metabolic mechanisms and obviating the need for metal-catalysed abiotic reductive aminations. This emerging sequence of prebiotic reactions could have set the stage for the advent of increasingly sophisticated pathways operating under catalytic control.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: The α-ketoacid pathway resembles transformations within the r-TCA and glyoxylate cycles.
Fig. 2: The α-ketoacid analogue pathway generated by the reaction of glyoxylate with pyruvate.
Fig. 3: The glyoxylate-dependent reduction of maloyl formate (III) to α-ketoglutarate (IV).
Fig. 4: Reaction progression of the α-ketoacid pathway with time.
Fig. 5: The transamination of α-ketoacids and glycine into α-amino acids and glyxoylate.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information.


  1. 1.

    Morowitz, H. J., Kostelnik, J. D., Yang, J. & Cody, G. D. The origin of intermediary metabolism. Proc. Natl Acad. Sci. USA 97, 7704–7708 (2000).

    CAS  PubMed  Google Scholar 

  2. 2.

    Huynen, M. A., Dandekar, T. & Bork, P. Variation and evolution of the citric-acid cycle: a genomic perspective. Trends Microbiol. 7, 281–291 (1999).

    CAS  PubMed  Google Scholar 

  3. 3.

    Meléndez-Hevia, E., Waddell, T. G. & Cascante, M. The puzzle of the krebs citric acid cycle: assembling the pieces of chemically feasible reactions, and opportunism in the design of metabolic pathways during evolution. J. Mol. Evol. 43, 293–303 (1996).

    PubMed  Google Scholar 

  4. 4.

    Nunoura, T. et al. A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science 563, 559–563 (2018).

    Google Scholar 

  5. 5.

    Zubarev, D. Y., Rappoport, D. & Aspuru-Guzik, A. Uncertainty of prebiotic scenarios: the case of the non-enzymatic reverse tricarboxylic acid cycle. Sci. Rep. 5, 8009 (2015).

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Wächtershäuser, G. Evolution of the first metabolic cycles. Proc. Natl Acad. Sci. USA 87, 200–204 (1990).

    PubMed  Google Scholar 

  7. 7.

    Cody, G. D. et al. Primordial carbonylated iron–sulfur compounds and the synthesis of pyruvate. Science 289, 1337–1340 (2000).

    CAS  PubMed  Google Scholar 

  8. 8.

    Cooper, G., Reed, C., Nguyen, D., Carter, M. & Wang, Y. Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites. Proc. Natl Acad. Sci. USA 108, 14015–14020 (2011).

    CAS  PubMed  Google Scholar 

  9. 9.

    Coggins, A. J. & Powner, M. W. Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis. Nat. Chem. 9, 310 (2017).

    CAS  PubMed  Google Scholar 

  10. 10.

    Mall, A. et al. Reversibility of citrate synthase allows autotrophic growth of a thermophilic bacterium. Science 359, 563–567 (2018).

    CAS  PubMed  Google Scholar 

  11. 11.

    Lehninger, A. L., Nelson, D. L. & Cox, M. M. Lehninger Principles of Biochemistry (WH Freeman, 2013).

  12. 12.

    Zubay, G. The glyoxylate cycle, a possible evolutionary precursor of the TCA Cycle. Chemtracts 16, 783–788 (2003).

    CAS  Google Scholar 

  13. 13.

    Peretó, J. Out of fuzzy chemistry: from prebiotic chemistry to metabolic networks. Chem. Soc. Rev. 41, 5394–5403 (2012).

    PubMed  Google Scholar 

  14. 14.

    Novikov, Y. & Copley, S. D. Reactivity landscape of pyruvate under simulated hydrothermal vent conditions. Proc. Natl Acad. Sci. USA 110, 13283–13288 (2013).

    CAS  PubMed  Google Scholar 

  15. 15.

    Zhang, X. V. & Martin, S. T. Driving parts of Krebs cycle in reverse through mineral photochemistry. J. Am. Chem. Soc. 128, 16032–16033 (2006).

    CAS  PubMed  Google Scholar 

  16. 16.

    Ralser, M. An appeal to magic? The discovery of a non-enzymatic metabolism and its role in the origins of life. Biochem. J 475, 2577–2592 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Sousa, F. L., Preiner, M. & Martin, W. F. Native metals, electron bifurcation, and CO2 reduction in early biochemical evolution. Curr. Opin. Microbiol. 43, 77–83 (2018).

    CAS  PubMed  Google Scholar 

  18. 18.

    Morowitz, H. J., Srinivasan, V. & Smith, E. Ligand field theory and the origin of life as an emergent feature of the periodic table of elements. Biol. Bull. 219, 1–6 (2010).

    CAS  PubMed  Google Scholar 

  19. 19.

    Keller, M. A., Piedrafita, G. & Ralser, M. The widespread role of non-enzymatic reactions in cellular metabolism. Curr. Opin. Biotechnol. 34, 153–161 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hartman, H. Speculations on the origin and evolution of metabolism. J. Mol. Evol. 4, 359–370 (1975).

    CAS  PubMed  Google Scholar 

  21. 21.

    Muchowska, K. B. et al. Metals promote sequences of the reverse Krebs cycle. Nat. Ecol. Evol 1, 1716–1721 (2017).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Pascal, R. A possible prebiotic basis for metabolism. Nature 569, 47–48 (2019).

    CAS  PubMed  Google Scholar 

  23. 23.

    Orgel, L. E. The implausibility of metabolic cycles on the prebiotic earth. PLoS Biol. 6, 0005–0013 (2008).

    CAS  Google Scholar 

  24. 24.

    Ross, D. S. The viability of a nonenzymatic reductive citric acid cycle—kinetics and thermochemistry. Orig. Life Evol. Biosph. 37, 61–65 (2007).

    CAS  PubMed  Google Scholar 

  25. 25.

    Kitadai, N., Kameya, M. & Fujishima, K. Origin of the reductive tricarboxylic acid (rTCA) cycle-type CO2 fixation: a perspective. Life 7, 39 (2017).

    PubMed Central  Google Scholar 

  26. 26.

    Maltais, T. R., VanderVelde, D., LaRowe, D. E., Goldman, A. D. & Barge, L. M. Reactivity of metabolic intermediates and cofactor stability under model early Earth conditions. Orig. Life Evol. Biosph. 50, 35–55 (2020).

  27. 27.

    Orgel, L. E. Self-organizing biochemical cycles. Proc. Natl Acad. Sci. USA 97, 12503–12507 (2000).

    CAS  PubMed  Google Scholar 

  28. 28.

    Springsteen, G., Yerabolu, J. R., Nelson, J., Rhea, C. J. & Krishnamurthy, R. Linked cycles of oxidative decarboxylation of glyoxylate as protometabolic analogs of the citric acid cycle. Nat. Commun. 9, 91 (2018).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Muchowska, K. B., Varma, S. J. & Moran, J. Synthesis and breakdown of universal metabolic precursors promoted by iron. Nature 569, 104–107 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Patel, B. H., Percivalle, C., Ritson, D. J., Duffy, C. D. & Sutherland, J. D. Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism. Nat. Chem. 7, 301–307 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Grabowski, J. J. Aqueous-phase pKa of the methyl group in acetic acid. Chem. Commun. 255–256 (1997).

  32. 32.

    Chiang, Y., Kresge, A. J. & Pruszynski, P. Keto–enol equilibria in the pyruvic acid system: determination of the keto–enol equilibrium constants of pyruvic acid and pyruvate anion and the acidity constant of pyruvate enol in aqueous solution. J. Am. Chem. Soc. 114, 3103–3107 (1992).

    CAS  Google Scholar 

  33. 33.

    Cooper, A. J. L., Ginos, J. Z. & Meister, A. Synthesis and properties of the α-keto acids. Chem. Rev. 83, 321–358 (1983).

    CAS  Google Scholar 

  34. 34.

    Buchanan, B. B. & Evans, M. C. W. The synthesis of alpha-ketoglutarate from succinate and carbon dioxide by a subcellular preparation of a photosynthetic bacterium. Biochemistry 54, 1212–1218 (1965).

    CAS  Google Scholar 

  35. 35.

    Ruffo, A., Testa, E., Adinolfi, A. & Pelizza, G. Control of the citric acid cycle by glyoxylate. 1. A new inhibitor of aconitase formed by the condensation of glyoxylate with oxaloacetate. Biochem. J. 85, 588 (1962).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Payes, B. & Laties, G. G. The inhibition of several tricarboxylic acid cycle enzymes by γ-hydroxy-α-ketoglutarate. Biochem. Biophys. Res. Commun. 10, 460–466 (1963).

    CAS  PubMed  Google Scholar 

  37. 37.

    Wiley, R. H. & Kim, K. S. The bimolecular decarboxylative self-condensation of oxaloacetic acid. J. Org. Chem. 38, 3582–3585 (1973).

    CAS  Google Scholar 

  38. 38.

    Herbst, R. M. & Engel, L. L. A reaction between alpha-ketonic acids and alpha-amino acids. J. Biol. Chem. 107, 505–512 (1934).

    CAS  Google Scholar 

  39. 39.

    Potter-McIntyre, S. L. & McCollom, T. M. Jarosite and alunite in ancient terrestrial sedimentary rocks: reinterpreting martian depositional and diagenetic environmental conditions. Life 8, 1–22 (2018).

    Google Scholar 

  40. 40.

    Wu, J. & Huang, S. X. Aluminum(iii) salts promoted transamination of glutamic acid for the synthesis of α-ketoglutaric acid. Chinese J. Org. Chem. 35, 1991–1993 (2015).

    CAS  Google Scholar 

  41. 41.

    Metzler, D. E., Ikawa, M. & Snell, E. E. A general mechanism for vitamin B6-catalyzed reactions. J. Am. Chem. Soc. 76, 648–652 (1954).

    CAS  Google Scholar 

  42. 42.

    Miller, S. L. Production of some organic compounds under possible primitive earth conditions. J. Am. Chem. Soc. 77, 2351–2361 (1955).

    CAS  Google Scholar 

  43. 43.

    Kvenvolden, K. et al. Evidence for extraterrestrial amino-acids and hydrocarbons in the Murchison meteorite. Nature 228, 923–926 (1970).

    CAS  PubMed  Google Scholar 

  44. 44.

    Wu, L.-F. & Sutherland, J. D. Provisioning the origin and early evolution of life. Emerg. Top. Life Sci. 3, 459–468 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Hammond, G. S. & Wu, C.-H. S. in Oxidation of Organic Compounds Vol. 77, 186–207 (American Chemical Society, 1968);

  46. 46.

    Theil, E. C. & Goss, D. J. Living with iron (and oxygen): questions and answers about iron homeostasis. Chem. Rev. 109, 4568–4579 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Mohammed, F. S. et al. A plausible prebiotic origin of glyoxylate: nonenzymatic transamination reactions of glycine with formaldehyde. Synlett 28, 93–97 (2017).

    CAS  Google Scholar 

  48. 48.

    Krishnamurthy, R. Life’s biological chemistry: a destiny or destination starting from prebiotic chemistry? Chem. Eur. J. 24, 16708–16715 (2018).

    CAS  PubMed  Google Scholar 

  49. 49.

    Yan, K. et al. Catalyst-free direct decarboxylative coupling of α-keto acids with thiols: a facile access to thioesters. Org. Biomol. Chem. 13, 7323–7330 (2015).

    CAS  PubMed  Google Scholar 

  50. 50.

    Bode, J. W., Fox, R. M. & Baucom, K. D. Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and alpha-ketoacids. Angew. Chem. Int. Ed 45, 1248–1252 (2006).

    CAS  Google Scholar 

Download references


This work was jointly supported by NSF and the NASA Astrobiology Program under the Center for Chemical Evolution (grant no. CHE-1504217) and by a NASA Exobiology grant to R.K. (80NSSC18K1300). G.S. acknowledges a Henry Dreyfus Teacher-Scholar Award.

Author information




R.T.S. and M.Y. contributed equally to this work. R.K., R.T.S and G.S. conceived the project. R.T.S, M.Y., R.K. and G.S. proposed and designed the experiments. R.T.S., G.S. and M.Y. carried out the experiments. All authors interpreted the data and discussed the experimental results. R.K. and G.S. supervised the research and wrote the paper with comments and feedback from R.T.S. and M.Y.

Corresponding authors

Correspondence to Ramanarayanan Krishnamurthy or Greg Springsteen.

Ethics declarations

Competing interests

R.K. and M.Y. declare they have no competing interests. G.S. and R.T.S. declare that a US non-provisional (16/746,124) and PCT application (PCT/US20/14023) have been filed covering the synthesis of organic acids and α-ketoacids. G.S. and R.T.S. own Aconabolics LLC, a company with commercial interests in using α-ketoacids as diagnostic agents.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–17.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Stubbs, R.T., Yadav, M., Krishnamurthy, R. et al. A plausible metal-free ancestral analogue of the Krebs cycle composed entirely of α-ketoacids. Nat. Chem. 12, 1016–1022 (2020).

Download citation

Further reading


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