Interestingly, while the elongation of loop 1 through the duplication of residues 13-15 (VVR) appears to be essential for the change of specificity towards the isomerization of PRA (facilitated by the presence of a new stabilizing arginine side chain close to the active sites, Figure , simply the loop duplication by itself is not enough to induce bifunctionality. That is, while the duplication elongates loop 1, it also rigidifies it, such that SeHisA(dup13-15) does show some ability to isomerize PRA (k cat > 0.15 s -1 ), but at the expense of losing all ability to isomerize the larger substrate ProFAR (no detectable activity). Therefore, the duplication by itself simply leads to a switch in activity from a modestly efficient isomerase towards ProFAR (k cat 7.8 s -1 ) towards a less efficient isomerase with activity towards PRA. Critical to the bifunctionality is the inclusion of an additional substitution, present in both variants studied above, namely D10G. This substitution increases the flexibility of the elongated loop 1 (Figure ), allowing for the loop to take on wide-open configurations which in turn facilitate the entry and binding of ProFAR to the active site (Figure , wide-open conformation). Our conventional MD simulations (10 x 500 ns per system) are overall short, taking in particular into account the slow turnover numbers of these enzymes (that suggest loop motions on the ms to s timescale). However, our observations that the D10G substitution leads to increased flexibility of loop 1 are in good agreement with prior NMR relaxation dispersion experiments. These detected μs to ms motions at 14 backbone 15 N positions in the SeHisA(dup13-15/D10G) variant, compared to only three positions for the SeHisA(dup13-15) variant, and with two resonances that are unique to SeHisA(dup13-15/D10G). As a result, adding this substitution is sufficient to convert SeHisA(dup13-15/D10G) back to a bifunctional enzyme, through exploitation of conformational dynamics, with k cat of 0.09 s -1 for the isomerization of PRA, and 0.05 s -1 for the isomerization of ProFAR (Table ). To explore this further, we therefore examined simulations of four loop elongated variants, specifically: dup13-15 (PDB ID: 5G2I 28 ), dup13-15/D10G (PDB ID: 5AC7, ), dup13-15/D10G/G102A (PDB ID: 5AC8 28 ), and dup13-15/D10G/G102A/Q24L/V15[b]M (PDB ID: