eliminase variants. To computationally probe the molecular origins for the enhanced chemical activities of the GNCA4-WT, GNCA4-12, V4-4 variants, as our starting point, we performed molecular dynamics simulations of each variant in complex with the substrate 5nitrobenzisoxazole, as described in the Experimental Section. In the case of the GNCA4-12 variant, we also performed simulations for its V4 variant, which presents an additional polypeptide segment, 20 in order to probe the impact of this polypeptide segment on catalysis. Analysis of these simulations give rise to two major observations, that align well with prior studies of de novo designed Kemp eliminases. The first of these (Figure ) is that the W290 side chain, which was inserted into the GNCA4-WT to improve stability and activity, is actually conformationally diverse and can take on a range of different side chain conformations. Upon optimization of the eliminase, in the V4-4 variant, we observe two discrete metastable conformations of the W290 side chain with a clear loss of a third (likely catalytically unfavorable) conformation of the tryptophan observed in GNCA4-WT. In this conformation, the tryptophan points out of the active site and away from the substrate (conformation I, Figure ). Importantly, the addition of the extra peptide from GNCA4-12 to V4 variant further results in a decrease in mobility of the W290 side chain, which might be related with the catalytic improvement of the enzyme. This is analogous to similar observations in the de novo designed Kemp eliminase KE07, where evolutionary conformational selection allowed for the emergence of multiple active site conformations (including multiple conformations of a key active site tryptophan) during directed evolution of this enzyme. However, in the case of KE07, this was a de novo active site based on a computational design involving a substantial number of mutations; in the current case, we now observe this effect on an enzyme with a de novo active site generated using a minimalist design involving a single mutation, that may mimic the actual emergence of new enzymes during natural evolution. The second crucial observation from our simulations is the conformational flexibility of the substrate (Figure ). Our de novo active site is built into a hydrophobic pocket originally evolved to stabilize a hydrophobic tryptophan side chain. While there is shape congruence between the substrate for Kemp elimination and the tryptophan side chain, the polar NO2 group of 5-nitrobenzisoxazole is unlikely to want to remain in this hydrophobic pocket. In fact, we see that the ligand gets pushed out of the active site for part of the simulation time, taking both an "IN" conformation which is similar to the conformation originally observed in the GNCA4-WT in complex with a transition state analogue, and an "OUT" conformation where the NO2 group points out of the hydrophobic pocket.