A. Loop Flexibility Enhancement
Several synthetic PETase lineages exhibited the spontaneous emergence of glycine-rich loops adjacent to the active site. These loops increased local flexibility, potentially facilitating:
Improved binding pocket accommodation
Enhanced substrate turnover
Reduced activation energy for hydrolysis
B. Allosteric Relay Formation
Another observed motif was the formation of long-range hydrogen bond relays between distal residues and the active site---an emergent allosteric control mechanism that modulated catalytic dynamics.
This suggests that our model captured non-local optimization, where distal mutations indirectly increased function---a feature rarely discovered through linear or site-specific mutagenesis.
C. -Sheet Rewiring and Barrel Formation
In some high-fitness branches, mutations led to the fusion of -strands, resulting in partial barrel-like motifs not seen in the original PETase structure. These barrels contributed to enhanced thermal stability and resistance to denaturation, essential traits for industrial biocatalysts operating in variable environments.
3. Evolutionary Cycles as Phase Transitions
