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.

  • Article
  • Published:

Cyanide as a primordial reductant enables a protometabolic reductive glyoxylate pathway

Nature Chemistry volume 14, pages 170–178 (2022)Cite this article

Subjects

Abstract

Investigation of prebiotic metabolic pathways is predominantly based on abiotically replicating the reductive citric acid cycle. While attractive from a parsimony point of view, attempts using metal/mineral-mediated reductions have produced complex mixtures with inefficient and uncontrolled reactions. Here we show that cyanide acts as a mild and efficient reducing agent mediating abiotic transformations of tricarboxylic acid intermediates and derivatives. The hydrolysis of the cyanide adducts followed by their decarboxylation enables the reduction of oxaloacetate to malate and of fumarate to succinate, whereas pyruvate and α-ketoglutarate themselves are not reduced. In the presence of glyoxylate, malonate and malononitrile, alternative pathways emerge that bypass the challenging reductive carboxylation steps to produce metabolic intermediates and compounds found in meteorites. These results suggest a simpler prebiotic forerunner of today’s metabolism, involving a reductive glyoxylate pathway without oxaloacetate and α-ketoglutarate—implying that the extant metabolic reductive carboxylation chemistries are an evolutionary invention mediated by complex metalloproteins.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Cyanide-mediated reduction via the addition–hydrolysis–decarboxylation process.
Fig. 2: Cyanide-mediated transformations of α-ketoacid analogues of TCA cycle produced from the reaction of glyoxylate with pyruvate.
Fig. 3: Reactions enabling a pathway that bypasses the α-ketoacids of the TCA cycle: oxaloacetate, α-ketoglutarate and pyruvate.
Fig. 4: Homologation of α-ketoacids using malononitrile and malonate as acetate equivalents.
Fig. 5: A hypothetical prebiotic reductive glyoxylate cycle as a forerunner to the r-TCA cycle.

Similar content being viewed by others

Data availability

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

References

  1. Wächtershäuser, G. Before enzymes and templates: theory of surface metabolism. Microbiol. Rev. 52, 452–484 (1988).

    Article  PubMed  PubMed Central  Google Scholar 

  2. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Nunoura, T. et al. A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science 359, 559–563 (2018).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Steffens, L. et al. High CO2 levels drive the TCA cycle backwards towards autotrophy. Nature 592, 784–788 (2021).

    Article  CAS  PubMed  Google Scholar 

  6. McMurry, J. & Begley, T. The Organic Chemistry of Biological Pathways (Roberts, 2005).

  7. Dolan, S. K. & Welch, M. The glyoxylate shunt, 60 years on. Annu. Rev. Microbiol. 72, 309–330 (2018).

    Article  CAS  PubMed  Google Scholar 

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

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Martin, W. F. Carbon–metal bonds: rare and primordial in metabolism. Trends Biochem. Sci. 44, 807–818 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Muchowska, K. B., Varma, S. J. & Moran, J. Nonenzymatic metabolic reactions and life’s origins. Chem. Rev. 120, 7708–7744 (2020).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  13. Guzman, M. I. & Martin, S. T. Photo-production of lactate from glyoxylate: how minerals can facilitate energy storage in a prebiotic world. Chem. Commun. 46, 2265–2267 (2010).

    Article  CAS  Google Scholar 

  14. Orgel, L. E. The implausibility of metabolic cycles on the prebiotic Earth. PLoS Biol. 6, e18 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ferris, J. P. & Hagan, W. J. Jr. HCN and chemical evolution: the possible role of cyano compounds in prebiotic synthesis. Tetrahedron 40, 1093–1120 (1984).

    Article  CAS  PubMed  Google Scholar 

  16. Sutherland, J. D. The origin of life—out of the blue. Angew. Chem. Int. Ed. 55, 104–121 (2016).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ritson, D. & Sutherland, J. D. Prebiotic synthesis of simple sugars by photoredox systems chemistry. Nat. Chem. 4, 895–899 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Coggins, A. J. & Powner, M. W. Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis. Nat. Chem. 9, 310–317 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Canavelli, P., Islam, S. & Powner, M. W. Peptide ligation by chemoselective aminonitrile coupling in water. Nature 571, 546–549 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Foden, C. S. et al. Prebiotic synthesis of cysteine peptides that catalyze peptide ligation in neutral water. Science 370, 865–869 (2020).

    Article  CAS  PubMed  Google Scholar 

  22. Eschenmoser, A. The TNA-family of nucleic acid systems: properties and prospects. Orig. Life Evol. Biosphere 34, 277–306 (2004).

    Article  CAS  Google Scholar 

  23. Butch, C. et al. Production of tartrates by cyanide-mediated dimerization of glyoxylate: a potential abiotic pathway to the citric acid cycle. J. Am. Chem. Soc. 135, 13440–13445 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yerabolu, J. R., Liotta, C. L. & Krishnamurthy, R. Anchimeric-assisted spontaneous hydrolysis of cyanohydrins under ambient conditions: implications for cyanide-initiated selective transformations. Chem. Eur. J. 23, 8756–8765 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bender, M. L. & Connors, K. A. A non-enzymatic olefinic hydration under neutral conditions: the kinetics and mechanism of the hydration of fumaric acid monoanion. J. Am. Chem. Soc. 84, 1980–1986 (1962).

    Article  CAS  Google Scholar 

  27. Colín-García, M., Negrón-Mendoza, A. & Ramos-Bernal, S. Organics produced by irradiation of frozen and liquid HCN solutions: implications for chemical evolution studies. Astrobiology 9, 279–288 (2009).

    Article  PubMed  Google Scholar 

  28. Keller, M. A., Kampjut, D., Harrison, S. A. & Ralser, M. Sulfate radicals enable a non-enzymatic Krebs cycle precursor. Nat. Ecol. Evol. 1, 0083 (2017).

    Article  PubMed Central  Google Scholar 

  29. Kunz, D. A., Chen, J.-L. & Pan, G. Accumulation of α-keto acids as essential components in cyanide assimilation by Pseudomonas fluorescens NCIMB 11764. Appl. Environ. Microbiol. 64, 4452–4459 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Eschenmoser, A. & Loewenthal, E. Chemistry of potentially prebiological natural products. Chem. Soc. Rev. 21, 1–16 (1992).

    Article  CAS  Google Scholar 

  33. Leopold, K. R. & Haim, A. Equilibrium, kinetics, and mechanism of the malonic acid–iodine reaction. Int. J. Chem. Kinet. 9, 83–95 (1977).

    Article  CAS  Google Scholar 

  34. Ghorai, S., Laskin, A. & Tivanski, A. V. Spectroscopic evidence of keto-enol tautomerism in deliquesced malonic acid particles. J. Phys. Chem. A 115, 4373–4380 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Mainguet, S. E., Gronenberg, L. S., Wong, S. S. & Liao, J. C. A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli. Metabol. Eng. 19, 116–127 (2013).

    Article  CAS  Google Scholar 

  36. Toney, M. D. Carbon acidity in enzyme active sites. Front. Bioeng. Biotechnol. https://doi.org/10.3389/fbioe.2019.00025 (2019).

  37. Wolfenden, R., Lewis, C. A. Jr & Yuan, Y. Kinetic challenges facing oxalate, malonate, acetoacetate, and oxaloacetate decarboxylases. J. Am. Chem. Soc. 133, 5683–5685 (2011).

  38. 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).

    Article  CAS  PubMed  Google Scholar 

  39. Cody, G. D. et al. Geochemical roots of autotrophic carbon fixation: hydrothermal experiments in the system citric acid, H2O–(±FeS)–(±NiS). Geochim. Cosmochim. Acta 65, 3557–3576 (2001).

    Article  CAS  Google Scholar 

  40. Varma, S. J., Muchowska, K. B., Chatelain, P. & Moran, J. Native iron reduces CO2 to intermediates and end-products of the acetyl-CoA pathway. Nat. Ecol. Evol. 2, 1019–1024 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Liu, Z. et al. Prebiotic photoredox synthesis from carbon dioxide and sulfite. Nat. Chem. 13, 1126–1132 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ritson, D. J. A cyanosulfidic origin of the Krebs cycle. Sci. Adv. 7, eabh3981 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Raulin, F., Bloch, S. & Toupance, G. Addition reactions of malonic nitriles with alkanethiol in aqueous solution. Orig. Life Evol. Biosph. 8, 247–257 (1977).

    Article  CAS  Google Scholar 

  45. Freeman, F. Chemistry of malononitrile. Chem. Rev. 69, 591–624 (1969).

    Article  CAS  PubMed  Google Scholar 

  46. Grönberg, K. L. C., Gormal, C. A., Durrant, M. C., Smith, B. E. & Henderson, R. A. Why R-homocitrate is essential to the reactivity of FeMo-cofactor of nitrogenase: studies on NifV-extracted FeMo-cofactor. J. Am. Chem. Soc. 120, 10613–10621 (1998).

    Article  Google Scholar 

  47. Fay, A. W. et al. Formation of a homocitrate-free iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase assembly. Dalton Trans. 39, 3124–3130 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Russell, J. B. & Forsberg, N. Production of tricarballylic acid by rumen microorganisms and its potential toxicity in ruminant tissue metabolism. Br. J. Nutr. 56, 153–162 (1986).

    Article  CAS  PubMed  Google Scholar 

  49. Sugimoto, N., Engelgau, P., Jones, A. D., Song, J. & Beaudry, R. Citramalate synthase yields a biosynthetic pathway for isoleucine and straight- and branched-chain ester formation in ripening apple fruit. Proc. Natl Acad. Sci. USA 118, e2009988118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Pizzarello, S. & Garvie, L. A. J. Sutter’s Mill dicarboxylic acids as possible tracers of parent-body alteration processes. Meteor. Planet. Sci. 49, 2087–2094 (2014).

    Article  CAS  Google Scholar 

  51. Pizzarello, S. & Shock, E. The organic composition of carbonaceous meteorites: the evolutionary story ahead of biochemistry. Cold Spring Harb. Perspect. Biol. 2, a002105–a002105 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Pizzarello, S. The chemistry of life’s origin: a carbonaceous meteorite perspective. Acc. Chem. Res. 39, 231–237 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Ball, R. & Brindley, J. The power without the glory: multiple roles of hydrogen peroxide in mediating the origin of life. Astrobiology 19, 675–684 (2019).

    Article  CAS  PubMed  Google Scholar 

  54. Springsteen, G., Rice, G., Yerabolu, J. & Krishnamurthy, R. The abiotic oxidation of organic acids to malonate. Synlett 28, 98–102 (2016).

    Article  Google Scholar 

  55. Lazcano, A. & Miller, S. L. On the origin of metabolic pathways. J. Mol. Evol. 49, 424–431 (1999).

    Article  CAS  PubMed  Google Scholar 

  56. Harrison, S. A. & Lane, N. Life as a guide to prebiotic nucleotide synthesis. Nat. Commun. 9, 5176 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  58. Chen, X., Li, N. & Ellington, A. D. Ribozyme catalysis of metabolism in the RNA world. Chem. Biodiver. 4, 633–655 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Mohammed, F. S. et al. A plausible prebiotic origin of glyoxylate: nonenzymatic transamination reactions of glycine with formaldehyde. Synlett 28, 93–97 (2016).

    Article  Google Scholar 

  61. Marin-Yaseli, M. R., Gonzalez-Toril, E., Mompean, C. & Ruiz-Bermejo, M. The role of aqueous aerosols in the “glyoxylate scenario”: an experimental approach. Chem. Eur. J. 22, 12785–12799 (2016).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a NASA (National Aeronautics and Space Administration) Exobiology grant 80NSSC18K1300 (R.K.) and jointly supported by National Science Foundation, the NASA Astrobiology Program under the Center for Chemical Evolution grant no. CHE-1504217 (R.K.) and a grant from the Simons Foundation 32712FY19 (R.K.). We thank A. Lazcano, G. Springsteen and L. Leman for feedback on the manuscript.

Author information

Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Mahipal Yadav, Sunil Pulletikurti & Ramanarayanan Krishnamurthy

  2. NSF-NASA Center for Chemical Evolution, Georgia Institute of Technology, Atlanta, GA, USA

    Sunil Pulletikurti & Ramanarayanan Krishnamurthy

  3. NCI at Frederick, Frederick, MD, USA

    Jayasudhan R. Yerabolu

Authors
  1. Mahipal Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sunil Pulletikurti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jayasudhan R. Yerabolu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ramanarayanan Krishnamurthy
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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

Corresponding author

Correspondence to Ramanarayanan Krishnamurthy.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks Juli Pereto and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Cyanide mediated reduction of oxaloacetate to malate.

Time course 1H-NMR of the reaction of oxaloacetate with cyanide (a) after 30 min of addition of cyanide to oxaloacetic acid and (b) after 120 days. The formation of the intermediates, cyanohydrin adduct of oxaloacetate and 2-carboxymalate 4b en route to malate 5 was observed. Traces of pyruvate 21 was formed from the competing decarboxylation reaction of oxalocetate.

Extended Data Fig. 2 Reduction of the conjugated double bonds of fumarate (derivatives) by cyanide.

(a) 1H-NMR (D2O) documenting the cyanide-mediated reduction of fumarate to succinate. The reduction of fumarate proceeds cleanly over days to produce predominantly succinate. (b) The reduction of fumaramide 7 (potentially derived from fumaronitrile) proceeds via the partially hydrolyzed intermediate fumaramate 8. The presence of cyanide at pH 9 first hydrolyzes 7 to the half amide 8, which then undergoes the addition of cyanide followed by hydrolysis and decarboxylation to form succinate 10. Slight shift of succinate peak in NMR spectrum (C, after 11 days) is due to change in pH.

Extended Data Fig. 3 Cyanide interaction with β-ketoglutarate leading to the formation of citrate and citramalic acid.

(Top) The reaction scheme showing the pathway for the formation of citrate 14 and citramalic acid 19b. (Bottom) Stacked time course 1H-NMR (D2O) spectra of the cyanide mediated reduction of β-ketoglutarate to citrate and formation of citramalic acid as a side product.

Extended Data Fig. 4 A cyanide catalyzed novel non-oxidative decarboxylation of a conjugated α-ketoacid 21 with simultaneous reduction.

(a, b) 1H-NMR time-course stacked spectra of the reaction of fumaroyl formate 21 (in equilibrium with hydroxy-2-ketoglutarate 20) with cyanide forms a cyanohydrin adduct that undergoes decarboxylation and concomitant reduction of the double bond to yield succinate 10, bypassing fumarate (which is the product formed from oxidative decarboxylation30 of 21).

Extended Data Fig. 5 Reaction between carboxysuccinate and glyoxylate leading to isocitrate.

1H-NMR (D2O) showing (top) the reaction mixture resulting from the reaction of carboxysuccinate 9 to isocitrate 12, which exists as the open-chain and closed (lactone) diastereomers. The bottom spectrum is of the mixture of 1:1 authentic threo-D/L-isocitrate 12 and its lactone 12a shown for comparison.

Extended Data Fig. 6 One-pot reaction of malonate with glyoxylate in the presence of cyanide leading to the formation of isocitrate.

Time course 1H-NMR of the reaction of malonate with glyoxylate (a) after 4 days which shows the formation of intermediates 16 and 30 and (b) after 35 days showing the formation of isocitrate isomers 12 and its lactone isomers 12a. See also Supplementary Figs. 69-70.

Extended Data Fig. 7 Reaction of malononitrile with α-ketoglutarate forming homocitrate.

1H-NMR spectra (a) of authentic homocitrate for comparison and (b) of the reaction mixture resulting from the reaction showing the formation of homocitrate 34. 11 = α-ketoglutarate; 28 = malonate. See also Supplementary Figs. 80-88.

Supplementary information

Supplementary Information

Supplementary Figs. 1–88 (1H and 13C NMR spectral data), materials and methods, procedures, experimental data, and references.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yadav, M., Pulletikurti, S., Yerabolu, J.R. et al. Cyanide as a primordial reductant enables a protometabolic reductive glyoxylate pathway. Nat. Chem. 14, 170–178 (2022). https://doi.org/10.1038/s41557-021-00878-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-021-00878-w

This article is cited by

Search

Advanced 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