Sulfate radicals enable a non-enzymatic Krebs cycle precursor
Markus A. Keller, Domen Kampjut, Stuart A. Harrison & Markus Ralser
Nature Ecology & Evolution 1, Article number: 0083 (2017)
Evolutionary theory Chemical origin of life
Received: 13 August 2016 Accepted: 13 January 2017
Published online: 13 March 2017
Abstract
The evolutionary origins of the Krebs cycle (tricarboxylic acid cycle) are not currently clear. Despite the existence of a simple non-enzymatic Krebs cycle catalyst being dismissed only a few years ago as ‘an appeal to magic’, citrate and other intermediates have since been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify a metabolism-like non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate compounds in a reaction mixture that orients on the typical components of Archaean sediment. Krebs cycle intermediates were found to be stable in water and in the presence of most molecule species, including simple iron sulfate minerals. However, in the presence of sulfate radicals generated from peroxydisulfate, the intermediates underwent 24 interconversion reactions. These non-enzymatic reactions covered the critical topology of the oxidative Krebs cycle, the glyoxylate shunt and the succinic-semialdehyde pathway. Assembled in a chemical network, the reactions achieved over 90% carbon recovery. Our results show that a non-enzymatic precursor of the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals.
The tricarboxylic acid (TCA) cycle, or Krebs cycle, is a central metabolic pathway, typically of oxidative function. This metabolic pathway provides precursors for the biosynthesis of amino acids, and plays an essential role in fatty-acid breakdown, cellular respiration, and energy and redox metabolism 1. The widespread occurrence of at least its oxidative reactions 2,3 indicates that at least parts of the Krebs cycle originated at a very early stage in evolution; perhaps dating back to the origin of life4,5,6. A frequently discussed hypothesis proposes that the Krebs cycle obtained its structural topology by Darwinian selection principles that were enabled by the ‘ribonucleic acid (RNA) world’. A post-genetic origin implies that pathway topologies are subject to progressive change, implying that the modern Krebs cycle could differ substantially from its early precursors 7. However, a post-genetic origin of metabolism struggles to explain how multiple, structurally complex enzymes came into being initially; enzymes themselves are made-up from the metabolic products of the Krebs cycle. Second, a Darwinian origin for the metabolic network topology has difficulties in explaining the high number of reactions that recur between kingdoms despite a lack of enzyme sequence conservation 8. An alternative hypothesis thus proposes that at least the key metabolic reactions originated from environmental chemistry. In this scenario, inorganic catalysis determines the basic structure of metabolism 9,10,11,12.
As enzymatic mechanisms of the Krebs cycle have limited resemblance to inorganic catalysis, the idea of a non-enzymatic origin was received sceptically by many 5,13. For instance, Leslie Orgel, a leading scientist in shaping the RNA world hypothesis, dismissed the possibility that a simple inorganic catalyst that could replace a series set of TCA-like reactions as ‘an appeal to magic’ 5. However, despite the fact that a simple catalyst was indeed missing, others have argued that several Krebs cycle metabolites form in organic chemical reactions 9,10. Meanwhile, the presence of a series of TCA intermediates has been confirmed on a carbonaceous meteorite 14. Furthermore, citrate and other TCA intermediates undergo highly efficient reductive interconversion reactions on semiconductor particles when exposed to strong ultraviolet light15. A non-enzymatic precursor to the Krebs cycle is therefore catalytically possible. Moreover, the existence of a unifying, simple catalyst has become plausible. Non-enzymatic reactions that replicate two other metabolic pathways, glycolysis and the pentose phosphate pathway, are united in their common dependence on ferrous iron as the catalyst and co-substrate 16,17. Fe(II) is abundant in typical Archaean sediments 18,19, implying that general chemical environments, rather than niche conditions, may have been key to shaping the structure of metabolic pathways.
We chose a systematic screening strategy whereby ~4,850 absolute quantitative experiments were performed to examine the reactivity of TCA intermediates in the presence of typical Archaean sediment constituents, as well as related iron and sulfur species. We found that the TCA intermediates were unreactive in the presence of the majority of iron and sulfate combinations, including the simple mineral, ferrous sulfide (FeS). However, in the presence of the radical donor peroxydisulfate, we detected 24 non-enzymatic interconversion reactions. These reactions resemble the isomerization and oxidative reactions of the enzymatic Krebs cycle, the glyoxylate shunt and the succinic semialdehyde pathway, so that their critical topologies are covered. A chemical network assembled from these reactions achieves more than 90% carbon recovery, forming a plausible non-enzymatic precursor for the origin of the early Krebs cycle.
Acknowledgements
We thank G. Averill and T. Littmann for helping with experiments. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001134), the UK Medical Research Council (FC001134) and the Wellcome Trust (FC001134). M.R. is supported by a Wellcome Trust grant, RG 093735/Z/10/Z, and a European Research Council Starting Grant, 260809. M.A.K. is supported by an Erwin Schrödinger postdoctoral fellowship (FWF, Austria, J3341). D.K. is supported by an Ad Futura studentship (Slovene Scholarship Fund).
Author information
Affiliations
Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
Markus A. Keller, Domen Kampjut, Stuart A. Harrison & Markus Ralser
Division of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria
Markus A. Keller
Division of Human Genetics, Medical University of Innsbruck, Peter-Mayr-Straße 1, 6020 Innsbruck, Austria
Markus A. Keller
The Molecular Biology of Metabolism Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
Markus Ralser
Contributions
M.A.K. and M.R. designed the research. M.A.K., D.K. and S.A.H. performed the research. M.A.K. and M.R. wrote the first draft of the paper, and all authors contributed to finalizing the manuscript.
Competing interests
The authors declare no competing financial interests.
Corresponding author
Correspondence to Markus Ralser.
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