Prior work on chalcone isomerase has shown that both substrate flipping and active site conformational dynamics are crucial for the evolution of CHI from a non-catalytic solute binding ancestor to extant proficient isomerases. Finally, laboratory evolution of the phosphotriesterase from Pseudomonas diminuta (PTE) caused an activity switch to an arylesterase; As can be seen from the scatter plots shown in Figures 8, S10 and S11, encouragingly, KIF is able to predict a large number of hotspot residues in all systems (38% in GNCA4-12, 70% in CHI and 42% in PTE), with KIF importance scores of >0.1. This is particularly encouraging in the case of PTE, where we see an importance score of >0.8 assigned to His254 in the initial R0 variant of this enzyme (basically, wild-type PTE), given that the mutation of His254 to arginine was the first mutation along the PTE evolutionary trajectory to fixate in the evolution towards arylesterase activity. KIF's ability to predict ~50% of experimentally known hotspots across these three systems is significant, both because one does not necessarily expect all hotspots to lie along residue communication networks, but also, because at this hit rate, KIF is able to predict key residues on which to focus directed evolution efforts, which is particularly valuable in systems where complete determination of NMR hotspots is not possible (for instance due to system size, as one example).