As Kemp elimination is an anthropogenic activity, for which we have engineered a de novo active site, we also wanted to test how well KIF would be able to predict hotspots in naturally occurring enzymes where key functionally important hotspots are known. To address this, we extended our KIF analysis to simulations of two additional systems: a chalcone isomerase ancestor inferred from ancestral sequence reconstruction (CHI ancCC, Figure ) and a bacterial phosphotriesterase from Pseudomonas diminuta (PTE, Figure ).