To determine the effects of the S/I double substitution on PETase dynamics, we analyzed the root mean square fluctuations (RMSF) of the H/F and S/I variants of BurPL, TfCut, LCC, and IsPETase, across a cumulative 2.5 μs of conventional MD simulations of each unliganded system (5 x 500 ns trajectories per system) at their respective temperature and pH optima. In all cases, comparison of the S/I and H/F variants did not show significant differences in RMSF throughout the protein (Figure ), except for the W-and D-loops (Figure ), which harbor the conserved active site tryptophan (W185 in IsPETase) and the catalytic aspartic acid (D206 in IsPETase), respectively. ). This could be explained by the longer extension of the H-loop in IsPETase and BurPL than in the thermophilic enzymes, which enables the formation of a continuous cleft for substrate binding. Despite the differences in loop flexibility of the key active site loops, two of which carry residues from the catalytic triad (Figure ), we observe only subtle differences in the hydrogen bonding patterns between the side chains of the catalytic serine (S160 in IsPETase) and histidine (H237 in IsPETase) and of the histidine and aspartate (D206 in IsPETase) for the S/I and H/F variants of each system (Figure ). It is worth noting that while the differences within each system are small, there is a more marked effect when comparing between different enzymes, and in particular between the mesophilic IsPETase and BurPL and the thermophilic TfCut and LCC enzymes. Here, all mesophilic enzymes exhibit substantially lower occupancy of the key hydrogen bonding interactions, and in particular the serine-histidine interaction (Figure ), than their thermophilic counterparts. Following from this, we also calculated the volume of the active site cleft in all enzymes using POVME 3.0. The resulting averages and standard deviations are shown in Table . As can be seen from this data, with the exception of BurPL, the H/F and S/I variants of each system have similar active site volumes and fluctuations. However, there is quite a spread in active site volume and flexibility (using standard deviation as a proxy measure for flexibility), with BurPL and TfCut having the most compact and least flexible clefts, and LCC having the largest and most flexible cleft. The cleft average volume and fluctuations depend on the dynamics of helices, sheets and loops forming the cleft itself, most importantly that of the residues whose side chain point inside the cleft itself.