Our results confirm the role for W145 that was proposed previously, based on experimentally determined structures. This is furthermore consistent with prior structural analysis that indicates that HisA activity is abolished in SeHisA(dup13-15), because the extended conformation of loop 1 blocks this side chain from interacting with ProFAR. In contrast, in the case of PriA, and again in agreement with prior structural analysis, we observe two possible conformations of loop 5, depending on which substrate is bound to the active site. That is, when ProFAR is bound to the active site, we sample a conformation similar to that observed in wild-type HisA (Figure ) with a similar stacking interaction between ProFAR and W145 (Figure ), however, the loop 5 rearrangement required to optimize the stacking position of W145 with ProFAR creates transient electrostatic repulsion between loop 5 and the rest of the enzyme, making this loop more conformationally dynamic, which we observe in our analysis in the form of an increased standard deviation in the distance and angle of the corresponding stacking interaction (d = 4.1 ± 0.6 Å, g = 19.2 ± 13.9°) (Figures and). The greater plasticity of this interaction, in turn, decreases substrate stability in the active site (the ProFAR RMSF increases from 18.4 Å in our simulations of wild-type HisA to 22.2 Å in our simulations of wild-type PriA), and thus the corresponding ProFAR isomerization activity of PriA.