For all enzymes, the conserved W-loop tryptophan is facing a tyrosine residue, forming an aromatic clamp into which one of the terephtalic rings of PET binds. When W185 in IsPETase (PDB ID: 5XG0 ) adopts conformer B, the distance between the Cγ-atoms of this residue and Y87 in the aromatic clamp is 9.2 Å, whereas for conformer C, the distance is 7.8 Å (Figure ). Therefore, we analyzed the change in distance between these aromatic residues in all MD simulations. As shown in Figure , a median distance of ~8.1 Å, similar to the experimental distance for the C conformer of W185, is obtained from the MD simulations for both IsPETase and BurPL. In contrast, a median distance of 8.9 Å, resembling the experimental distance for conformer B, is observed for simulations of IsPETase and BurPL with the S/I substitutions. In contrast, no differences are observed for the median distance of both variants of TfCut and LCC, with TfCut having a median distance 2 Å away from the crystallographic distance (PDB ID: 5ZOA ), whereas LCC is closer to the experimental distance (PDB ID: 4EB0 26 ) (Figure ). a All values were calculated using POVME 3.0, and are averages and standard deviations over 5 x 500 ns conventional MD simulations per system, with snapshots taken every 2.5 ns of simulation time (2,000 frames analyzed per system). Following from this, we performed shortest path map (SPM) analysis on the MD replicas to analyze the allosteric communication pathways that potentially connect the different active site loop regions. The SPM method allows for the calculation of the shortest pathways between all residues (the nodes) in the network. Nodes and edges (pathways between nodes) that are often used for communication between residues can then be identified, and these residues can therefore be considered as being important for regulating the global conformational dynamics of the enzyme. The calculated allosteric communication pathways are presented in Figure and illustrate the differences between enzymes from different source organisms. Loop D and the mutation-hosting helix are only mildly important for the pathway of both variants of IsPETase. In BurPL, loop H harbors significant residues whose motions are highly correlated.