Extended Data Fig. 2: Enzymes catalyzing reactions before the addition of ammonia are not depleted in nitrogen-containing amino acids relative to enzymes added after ammonia.

To see if the amino acid biases in active sites of enzymes catalyzing reactions added to the network without ammonia (see Extended Data Fig. 1e, f) is confounded due to evolutionary selection for reduced nitrogen in these enzymes, we computed the fraction of nitrogen side-chains in enzymes in pre-ammonia reactions (x-axis) and in enzymes in post-ammonia reactions (y-axis). We found that enzymes in the pre-ammonia networks did not have significantly less nitrogen usage compared to enzymes in post-ammonia reactions (one-tailed Wilcoxon signed-rank test: P = 1).