Structures and mutagenesis experiments have identified two key active site side chains in HisA and PriA, which are important for binding of the substrate ProFAR. These are W145, which forms a stabilizing stacking interaction with the substrate, and R83 (R85), which interacts with the second phosphodianion group of the substrate (Figure ). To further explore the conformational diversity of these key tryptophan and arginine residues in HisA and PriA, respectively, we examined the joint dihedral angle distribution of the side chains of these residues in simulations of unliganded HisA and PriA, as well as HisA and PriA in complex with both From these data it can be seen that both the tryptophan and arginine side chains are highly conformationally flexible in the unliganded enzymes. However, while the binding of the substrate ProFAR to HisA restricts the conformational space of the arginine side chain on the "gripper" loop 3 to a catalytically competent position that helps stabilize the bound substrate, in PriA, the R143 side chain is only 4.8Å from the "gripper" residue R85 (distance between the two side chain carbon atoms, based on PDB ID: 3ZS4 ). This in turn creates electrostatic repulsion between the two arginine side chains, thus destabilizing loop 5 as well as the interaction between the substrate ProFAR and the R85 side chain (Figure ). In contrast, when PRA, that lacks the second phosphodianion group, is bound to the PriA active site, the R83/R85 side chains increase their conformational flexibility again, sampling more or less the same conformational space as in the case of the unliganded enzyme (Figure ). Therefore, the interaction of these residues with the larger substrate ProFAR is likely playing an important role in the ability of these enzymes to bind and isomerize this compound.