A plausible metal-free ancestral analogue of the Krebs cycle composed entirely of α-ketoacids

Abstract

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.

Figure 1.

The α-ketoacid pathway (inner green arrows) resembles transformations within the r-TCA (black) and glyoxylate (purple) cycles. Starting from pyruvate (I) or oxaloacetate (VIII), under mild aqueous conditions, the α-ketoacid pathway requires only the addition of glyoxylate (II), acting as both the carbon source and reductant, to progress through a series of α-ketoacid analogs of metabolic carboxylate intermediates. The glyoxylate cycle in modern biology similarly uses acetyl-CoA (in place of pyruvate) and glyoxylate as building blocks. Although likely a later evolutionary invention, intermediates of the α-ketoacid pathway can be transformed into their respective TCA cycle intermediates via oxidative decarboxylation in the presence of an appropriate oxidant (peach arrows).

Figure 2.

The α-ketoacid analog pathway generated by the reaction of glyoxylate with pyruvate. (a) The reaction scheme highlights the pyruvate scaffold (in teal), a secondary pathway to α-ketoglutarate occurs through a di-DHKG intermediate (Supplementary Fig. 1), (b) Reaction yields from pyruvate and from α-ketoglutarate were determined by ¹H-NMR integration versus an internal t-BuOH standard and by HPLC-UV (at 210 nm) versus a standard curve. (C) ¹H NMR (in D₂O) of a 100 μl aliquot from a reaction of 200 mM pyruvate (I) and 3 equiv. of glyoxylate (II) in a 0.5 M phosphate buffer at pH 7 heated to 50 °C for 21 h, yields 39% maloyl formate (III), 6% fumaroyl formate (IV), 5% α -ketoglutarate (α -k.g., V), 5% isocitroyl formate (VI), 2% aconitoyl formate (VII) with 1% pyruvate remaining. (d) ¹H NMR (in D₂O) of a 100 μl aliquot from a reaction of 200 mM α-ketoglutarate (V) and 1 equiv. of glyoxylate (II) in a 0.5 M phosphate buffer at pH 7 heated to 50 °C for 21 h, yields 36% isocitroyl formate (VI) and 21% aconitoyl formate (VII) with 37% α-ketoglutarate remaining. Erythro (e) and threo (t) diastereomers of isocitroyl formate were identified by oxidation to isocitrate standards.

Figure 3.

The glyoxylate-dependent reduction of maloyl formate (III) to α-ketoglutarate (IV). (a) Two reaction pathways for the reduction of III to α-ketoglutarate (V) proceed through either IV (via a cross-Cannizzaro type hydride transfer to V), or di-DHKG (via decarboxylation and retro-Claisen fragmentation to V). Oxalate and formate side products formed from glyoxylate (highlighted in amber) provide evidence for the operation of both unique pathways. (b) ¹H NMR (in D₂O) of a 100 μl aliquot from a control reaction of 200 mM maloyl formate (III) in a 0.5 M pH 7 phosphate buffer heated to 50 °C for 21 h, (c) ¹H NMR (in D₂O) of a 100 μl aliquot from a reaction of 200 mM maloyl formate (III) and 1 equiv. glyoxylate (II) in a 0.5 M pH 7 phosphate buffer heated to 50 °C for 21 h. The maloyl formate (4-hydroxy-2-ketoglutarate, HKG) was obtained from a commercial source.

Figure 4.

Reaction progression of the α-ketoacid pathway with time. (a) The progression of the pyruvate and glyoxylate reaction sequence was visualized by an HPLC time course study after oxidation of reaction aliquots. 200 mM pyruvate with 3 equiv. of glyoxylate in a 0.5 M pH 7 phosphate buffer was heated to 50 °C for 1, 2, 3, 4, 6, 10, 14, 20, 27, 31, 35, 53, and 72 h, to yield 5.9% fumaroyl formate (IV) and 0.8% aconitoyl formate (VII) after 27 h, and 1.9 % fumarate and 2.3% aconitate after 72 h. Reaction aliquots were oxidized to the canonical r-TCA intermediates with the addition of 5 equiv. H₂O₂ stirred for 0.5 h at rt before analysis by HPLC/UV in comparison to seven-point standard calibration curves of the canonical standards. (b) ¹H NMR (in D₂O) of a 100 μl aliquot from a reaction of 200 mM pyruvate and 3 equiv. of glyoxylate in a 0.5 M pH 7 phosphate buffer heated to 50 °C for 21 h, followed by the addition of 5.0 equiv. of H₂O₂ and stirring for 0.5 h at rt. Erythro (e) and threo (t) diastereomers of isocitrate are labeled. (c) ¹H NMR (in D₂O) of a 100 μl aliquot from a reaction of 200 mM α-ketoglutarate and 1 equiv. of glyoxylate in a 0.5 M pH 7 phosphate buffer heated to 50 °C for 21 h, followed by the addition of 5.0 equiv. of H₂O₂ and stirring for 0.5 h at rt.

Figure 5.

The transamination of α-ketoacids and glycine into α-amino acids and glyxoylate. (a) α-ketoglutarate (V) and pyruvate (I) produce glutamate and pyruvate respectively in the presence of glycine as the nitrogen source, (b) ¹H NMR (in D₂O) of a reaction of 350 mM glycine with 1.2 equiv. of α-ketoglutarate in a pH 5 aqueous solution with 0.1 equiv. of AlK(SO₄)₂ heated to 80 °C yields 44% glutamate after 4 h, (c) an equivalent reaction of glycine with pyruvate yields alanine in 19% yield after 24 h.