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Coenzymes in a pre-enzymatic metabolism
Coenzymes in a pre-enzymatic metabolism
https://orcid.org/0009-0005-6089-5277
Dherbassy Quentin Conceptualization Investigation Project administration Visualization Writing - original draft Writing - review & editing 1
https://orcid.org/0000-0002-9864-7042
Mayer Robert J. Conceptualization Validation Visualization Writing - review & editing 2
https://orcid.org/0000-0002-7851-6133
Moran Joseph Conceptualization Funding acquisition Project administration Supervision Writing - review & editing 1 3 *
1 Institut de Science et d’Ingénierie Supramoléculaires (ISIS), CNRS UMR 7006, Université de Strasbourg - 8 Allée Gaspard Monge, 67000 Strasbourg, France.
2 Technische Universität München, School of Natural Sciences, Lichtenbergstr. 4, 85748 Garching, Germany.
3 Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
* Corresponding author. Email: jmoran@uottawa.ca
20 9 2024
18 9 2024
10 38 eadr535705 7 2024
12 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
2024
The Authors
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Experiments now support theoretical suggestions that coenzymes mediated key metabolic reactions before the emergence of enzymes. Three coenzymes believed essential to the core metabolism of the last universal common ancestor to extant life (pyridoxal phosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide) were recently found to be active in their corresponding metabolic reactions in the absence of enzymes. These findings suggest an earlier contribution of coenzymes to abiogenesis, ultimately yielding insights into the prebiotic origins of metabolism.

Recent experimental work suggests that coenzymes mediated key metabolic reactions before the emergence of enzymes.

http://dx.doi.org/10.13039/100010663 H2020 European Research Council 101001752 http://dx.doi.org/10.13039/501100001663 Volkswagen Foundation 96_742 http://dx.doi.org/10.13039/501100001804 Canada Research Chairs German Research Foundation MA 9687/1-1 Liebig fellowship by the Fonds der chemischen Industrie
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pmcINTRODUCTION

Coenzymes are involved in every core metabolic pathway, where they help proteins catalyze reactions that would otherwise be challenging for the limited chemical toolbox provided by amino acids. Thus, coenzymes are essential to life, dating back beyond the Last Universal Common Ancestor (LUCA), some 4 billion years ago (1). Coenzymes also represent one of the many “chicken-and-egg” paradoxes linked to the origins of life: The biosynthesis of coenzymes relies on proteins that, themselves, rely on coenzymes to function. How did this circular relationship begin? Bioinformatics analysis suggests that with as little as eight coenzymes, nearly a quarter of the metabolism of ancient organisms becomes possible (2). Gaining insight into how coenzymes became relevant to prebiotic chemistry should illuminate our understanding of the origins of metabolism and, more generally, life.

DISCUSSION

Coenzymes operate within the active sites of enzymes and are directly involved in making or breaking bonds. They, thus, may have operated before the advent of complex macromolecules. Supporting this idea, some coenzymes were known to exhibit limited activity in the absence of proteins, and the scaffolds of certain coenzymes are accessible under prebiotic conditions (3). However, the lack of reported nonenzymatic activity of several key coenzymes has, until recently, restrained the hypothesis that coenzymes once reinforced the prebiotic reactions network constituting protometabolism. Coenzymes were therefore restricted to a later stage of prebiotic chemistry, where protometabolism evolved into a complex system of autonomous reactions (4). Here, we highlight recent experimental studies from our laboratory that show that three of the most important coenzymes might indeed be capable of functioning in a pre-enzymatic stage of metabolism and thus could have made an early contribution in abiogenesis.

The coenzyme pyridoxal phosphate (PLP) is involved in the metabolism of all amino acids and, thus, is among those coenzymes with the largest impact on putative prebiotic networks (2). Since the 1950s, it has been recognized that PLP can participate in transamination reactions between α-keto acids and an amino group donor in the absence of enzymes, a reaction sometimes improved by the addition of metals. In 2023, Dherbassy et al. (5) showed that PL(P), when paired with a metal ion as a cocatalyst, is a more potent catalyst for transamination reactions than previously thought, enabling rate accelerations of >1000-fold and up to 7.9 catalyst turnovers. The reaction occurred under a wide range of pH, temperature, and required relatively low catalysts loading (5 mol %) (Fig. 1A). The improvement was most notable when PL(P) was paired with the most abundant metals in the earth’s crust, aluminum and iron (Fe3+ and Al3+), outcompeting catalysis by metals alone, even rarer ones. Mechanistic studies showed that the addition of metals modified the rate-determining step from deprotonation of the PL(P) amino acid adduct to the product-releasing hydrolysis of the ketimine formed in this very step. Lewis acidic metals thus facilitated deprotonation and stabilized the key imine intermediates against hydrolysis by 259-fold. This strong stabilization does, however, limit the catalyst’s turnover frequency by slowing down product release. PLP-catalyzed transamination would thus ideally operate in the absence of metals, as it has evolved to do in biology today by using enzymes, rather than metals, as cocatalysts. Thus, the observed synergy between PL(P) and metal ions can be interpreted as a potential intermediary stage of PLP-catalyzed transamination during the evolution of prebiotic chemistry into biochemistry.

Fig. 1. Experimental work describing the prebiotic activity of coenzymes central to metabolism.

(A) Pyridoxal-5′-phosphate (PLP) as a catalyst in transamination reactions, Dherbassy et al. (5). (B) Adenosine diphosphate (ADP)–promoted phosphorylation of nucleoside phosphates, Werner et al. (7). (C) Nicotinamide adenine dinucleotide hydride (NADH) in the reduction of α-keto acids to α-hydroxy acids, Mayer et al. (9)

The adenosine diphosphate (ADP)/adenosine triphosphate (ATP) couple is the most commonly used coenzyme in reconstructions of LUCA’s biochemistry, owing to its widespread role of shuttling phosphoryl groups within metabolism (2). However, the role these nucleotides might have played in pre-enzymatic metabolism remains unclear. Of the various classes of metabolites bearing phosphoryl groups, why did nature pick nucleotides to shuttle them around, and why, specifically adenosines, for most of metabolism? Nonenzymatic Fe3+-dependent phosphorylation of ADP to ATP using the biological phosphoryl donor acetyl phosphate was first reported in the 1990s, but in 2022, the same conditions were found not to phosphorylate nucleoside diphosphates bearing the other canonical nucleobases (6). In 2023, Werner et al. (7) showed that ADP not only promotes its own phosphorylation by acetyl phosphate but also the phosphorylation of other canonical and noncanonical nucleoside diphosphates (NDPs) to form nucleoside triphosphates (NTPs) under mild conditions (Fig. 1B). NTPs are especially important for the emergence of metabolism since only triphosphorylated nucleosides are capable of polymerizing to RNA. The adenine nucleobase and the formation of NDP-metal complexes with Fe3+ or Al3+ ions, observed by nuclear magnetic resonance and high-resolution mass spectrometry, were found to be essential in this nonenzymatic process. The N-7 of the imidazole-moiety of ADP was found to play a key role (Fig. 1B), reminiscent of the enzymatic phosphorylation of NDPs nowadays catalyzed by histidine residues. This finding suggests that nature’s choice of the ADP/ATP couple as a phosphoryl transfer catalyst in much of metabolism is no accident but may rather stem from its unique pre-enzymatic ability to promote phosphoryl group transfer from acyl phosphates to nucleotides.

Nicotinamide adenine dinucleotide (NAD), a modified ribonucleotide that exists in reduced (NADH) and oxidized (NAD+) forms, is life’s premier redox coenzyme and is believed to have upheld a similar status in LUCA and at the origin of metabolism (2). In conflict with this view, NADH was, until recently, thought to be unreactive to its biological substrates in the absence of enzymes (8, 9). However, studying the reactions of keto acids with NADH, Mayer et al. (9) found that catalytic amounts of the ions of abundant metals, especially Fe3+ and Al3+, enabled nonenzymatic reductions of keto acid. Thus, the reduction of several key metabolic α-keto acids to their corresponding α-hydroxy acids could be performed under mild conditions (Fig. 1C), tolerating high dilution (1 mM) and relatively low catalyst loading (5 mol %). Kinetic, mechanistic, and computational studies clarified the multiple roles of metal ions. Coordination of the metal with the keto acid increased the reactivity of the latter by more than six orders of magnitude. The metal also binds the coenzymes’s pyrophosphate moiety, yielding a 1:1:1 complex where the reaction proceeds via a favorable intramolecular pathway. The pre-organization of substrates led to chiral induction from the NADH ribose moieties to furnish D-hydroxy acids in 15 to 60% ee, a rare example where chiral information is relayed within a nonenzymatic metabolic reaction. As with PL(P), the metal ion–catalyzed reduction with NADH should be viewed as a potential earlier stage within a nonenzymatic metabolism, and the exploration of minimal biopolymers that might promote this reaction with higher selectivity is an exciting avenue for future research.

Collectively, these experimental studies substantiate theoretical models derived from the structure of metabolic networks, concluding that coenzymes could have participated in a protometabolism and performed their functions even before they had help from complex protein partners. To further support this hypothesis, it will be paramount to find more efficient prebiotic syntheses of coenzymes essential to early metabolic networks. The scaffolds of numerous coenzymes, including two of the three reviewed here, contain nucleotide moieties, linking them to the molecules of genetics (10). A common point among all three experimental studies is the reliance on cocatalysis by ions of the two most common metals in the Earth’s crust, aluminum and iron (Fe3+ and Al3+). Metals can fulfill multiple roles, acting as Lewis acids or pre-organizing the substrates and reactants, a function reminiscent of enzymes. To bridge the gap in the molecular evolution of coenzymes, one of the most promising areas of future research will be to uncover how uncoded peptides or simpler coded proteins took over cocatalysis from the inorganic environment, bridging prebiotic and biotic worlds. Shedding light on this question, Yu et al. (11) recently demonstrated that PL(P) and enantiopure peptides copromote stereoselective transamination reactions, setting the stereochemistry of amino acids. Using PL as a reagent, they were able to induce enantioselectivity in transamination half-reactions under acid-base catalysis from enantiopure peptides, with up to 50% ee at pH 8.5 and room temperature (Fig. 1A). Although catalytic turnover was not observed, this work nonetheless foreshadows another potential intermediary step in the molecular evolution of PL(P) catalysis. In the years ahead, it will be interesting to ascertain how the complexity of the peptide cocatalyst affects the efficiency and selectivity of a variety of coenzyme-mediated metabolic reactions. A deep understanding of biochemistry depends on it.

Acknowledgments

Funding: This work was supported by the German Research Foundation grant MA 9687/1-1 (R.J.M.), European Research Council (ERC) grant 101001752 (J.M.), Volkswagen Foundation grant 96_742 (J.M.), Canada Research Chair (J.M.), and Liebig fellowship by the Fonds der chemischen Industrie (R.J.M.).

Author contributions: J.M. supervised the project. Q.D., R.J.M., and J.M. conceptualized the project. Q.D. wrote the original draft. Q.D., R.J.M., and J.M. reviewed and edited the manuscript.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper.
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