In addition, in contrast to HisA, which undergoes conformational changes of loop 1 during the real-time evolution experiment that changes its selectivity from ProFAR-specific to PRAspecific, the bifunctional enzyme PriA is already able to rearrange its active site in its wildtype form, to accommodate the different substrates through alternation between Pro-ProFAR and Pro-PRA conformations of loop 5. These conformational changes both reduce repulsion between the two active site arginine side chains that in turn destabilize loop 5 dynamics (Figure ), as well as disrupting the stacking interaction between the W145 side chain and the substrate ProFAR (Figure ). This rationalizes the preference of this enzyme towards PRA rather than ProFAR, despite its similarities with the HisA active site, and we note also that loss of the stacking interaction between W145 and the substrate ProFAR was also presented as one aspect of the gain of PRA-isomerization activity in the SeHisA(dup13-15) variant from the real time evolution experiment. Finally, we performed EVB simulations of the first ring-opening step of ProFAR and (where relevant) PRA isomerization (Figure ) by wild-type HisA, PriA and variants. As described above, the actual rate of the chemical step in these enzymes is unknown, since the rate-limiting step is likely to occur off the enzyme. However, of the steps that occur in the enzyme active site, this is the step that is likely to be the slowest, and therefore it is of interest how the substitutions affect the rate of the ring-opening reaction. Here, we see that our calculated activation free energies for the ring-opening reaction trend well with the differences in activation free energies derived using the measured turnover numbers as an upper limit for this value, suggesting there is both a chemical and a dynamical component to the observed changes in activity upon substitution and/or duplication of key residues. Furthermore, in order to reproduce the relevant PriA activation free energies, it was necessary to start from different structures of loop 5, following earlier structural analysis that demonstrate the loop can exist in either a knot-like pro-PRA or beta-hairpin pro-ProFAR conformation (Figure ), depending on what product is bound to the active site. Clearly, the ease with which this rearrangement can occur will also impact the selectivity of this enzyme. Also, EVB simulations of wild-type SeHisA, MtPriA and the SeHisA(dup13-15/D10G/G102A/Q24L/V15[b]M) variant with loop 1 in an open conformation all yield substantially higher energies for PRA isomerization, whereas ProFAR isomerization (by wild-type HisA) seems to be unaffected (Tables and). This further emphasizes the importance of the correct closure of loop 1 for isomerization of the smaller substrate PRA. This is in contrast to substrate binding, where the conformational plasticity of loop 1 appears to be far more important for facilitating correct binding of ProFAR than PRA (Figure ).