The TIM-barrel structures of SeHisA and MtPriA are similar, with an RMSD of 1.09 Å between them (comparing the Ca atoms in PDB IDs 3ZS4 and 5A5W ). TmTrpF, in contrast, is a smaller enzyme with 40 fewer residues in the sequence than MtPriA and SeHisA. We performed 10 x 500 ns conventional molecular dynamics simulations of unliganded HisA, TrpF and PriA, and calculated the average and standard deviations of the active site volumes of each enzyme using the MDpocket 78 tool, which is provided as part of the fpocket 79 suite of pocket detection programs, as described in the Supporting Information. The resulting calculated volumes are shown in Table . We obtained average volumes of 745.4 ± 129.6 Å 3 , 1033.8 ± 217.0 Å 3 and 1173.5 ± 158.0 Å 3 , for the active sites of TrpF, PriA and HisA, respectively during our simulations. From this, it can be seen that the TrpF active site is more compact than that of PriA and HisA, which have successively larger active site volumes, with more "flexible" pockets than TrpF (using the standard deviation on the volume as a proxy for this flexibility). For comparison, the substrates ProFAR and PRA have volumes of 829 and 559 Å 3 , respectively, calculated using Alexander Balaeff's Mol_Volume program Version 1.0, with default radii of 1.7Å and a probe sphere of 0.5Å. This confirms the structural data indicating that the active site pocket of TrpF is too compact to accommodate the much larger substrate, ProFAR, leading to the selectivity of this enzyme towards PRA. Furthermore, the PriA active site is the most flexible of the three, in line with structural data that indicates that PriA is capable of significantly rearranging its active site (in particular loop 5 conformation) when accommodating the different substrates ProFAR and PriA (Figure ).