Extended Data Fig. 1: Enzymes in thioester-driven protometabolism are depleted in nitrogenous compounds.

(a) We classified reactions in KEGG as being plausibly pre-enzymatic (PPE) reactions if they (i) could proceed spontaneously, (ii) were associated with enzymes that contain at-least one iron–sulfur cluster or (iii) were associated with an enzyme that relied on atleast one metal (Ni, Co, Cu, Mg, Mn, Mo, Zn, Fe, W) cofactor. (b) For all scenarios resulting in expansion of  more than 100 metabolites (n = 144) we computed the fraction of PPE-reactions amongst the pre-ammonia reactions (x-axis) and post-ammonia reactions (y-axis). The frequency of PPE-reactions in the pre-ammonia reaction set was on average higher than the frequency of PPE-reactions in the post-ammonia reaction set (one-tailed Wilcoxon signed-rank test: P < 10^-19). (c) We identified KEGG reactions that were dependent on at-least one of the following nitrogen-containing coenzymes: flavin, biotin, thiamine pyrophosphate (TPP) pyridoxal phosphate (PLP), haem, pterin or cobalamin. (d) We compute the fraction of pre- and post- ammonia reactions associated with nitrogen-containing coenzymes in the KEGG database, and found that a much higher proportion of post-ammonia reactions were dependent on these coenzymes relative to pre-ammonia reactions (one-tailed Wilcoxon signed-rank test: P < 10^-24). (e) We parsed the catalytic active site database⁷² to find entries associated with pre and post-ammonia reactions, and compute the fraction of entries associated with amino acids with nitrogen-containing side-chains (Q,N,W,H,K,R). (f) For each scenario, the fraction of active sites with nitrogen-containing amino acids was significantly higher for post-ammonia reactions relative to pre-ammonia reactions one-tailed Wilcoxon signed-rank test: P < 10^-24).