In the case of W145, this side chain slightly increases its conformation flexibility when the smaller substrate PRA is bound to HisA (note that PRA was placed manually in the active site by overlay with substrate ProFAR, as described in the Supporting Information). However, in the case of PriA, both W145 the and R143 side chains are conformationally restricted to a catalytically competent position due to a rearrangement of loop 5 upon PRA binding, that swaps the position between these two residues compared to when ProFAR is bound to PriA, preventing the electrostatic repulsion between the R85 and R143 side chains that is observed when ProFAR (PRFAR) is bound to the active site (Figure ). As noted previously, the R83/R85 side chain is one of a number of key residues on the "gripper" loop that interact with the distal phosphodianion group of the larger substrate ProFAR, contributing to the stabilization of the substrate in HisA active site. Hence, in PriA, the loop 5 rearrangement required for ProFAR substrate binding prevents R85 from gripping the second phosphodianion group and thus showing clear preference for the isomerization of PRA substrate.