Structure and Energetics\nof PET-Hydrolyzing Enzyme\nComplexes: A Systematic Comparison from Molecular Dynamics Simulations

Discovered in 2016, the enzyme PETase, secreted by bacterial Ideonella Sakaiensis 201-F6, has an excellent hydrolytic\nactivity toward poly(ethylene terephthalate) (PET) at room temperature,\nwhile it decreases at higher temperatures due to the low thermostability.\nMany variants have been engineered to overcome this limitation, which\nhinders industrial application. In this work, we systematically compare\nPETase wild-type (WT) and four mutants (DuraPETase, ThermoPETase,\nFastPETase, and HotPETase) using standard molecular dynamics (MD)\nsimulations and unbinding free energy calculations. In particular,\nwe analyze the enzymes’ structural characteristics and binding\nto a tetrameric PET chain (PET4) under two temperature conditions: <i>T</i>1300 K and <i>T</i>2350 K. Our\nresults indicate that (i) PET4 forms stable complexes with the five\nenzymes at room temperature (∼300 K) and (ii) most of the interactions\nare localized close to the active site of the protein, where the W185\nand Y87 residues interact with the aromatic rings of the substrate.\nSpecifically, (iii) the W185 side-chain explores different conformations\nin each variant (a phenomenon known in the literature as “W185\nwobbling”). This suggests that the binding pocket retains structural\nplasticity and flexibility among the variants, facilitating substrate\nrecognition and localization events at moderate temperatures. Moreover,\n(iv) PET4 establishes aromatic interactions with the catalytic H237\nresidue, stabilizing the catalytic triad composed of residues S160-H237-D206,\nand helping the system achieve an effective configuration for the\nhydrolysis reaction. Conversely, (v) the binding affinity decreases\nat a higher temperature (∼350 K), retaining moderate interactions\nonly for HotPETase. Finally, (vi) MD simulations of complexes formed\nwith poly(ethylene-2,5-furan dicarboxylate) (PEF) show no persistent\ninteractions, suggesting that these enzymes are not yet optimized\nfor binding this alternative semiaromatic plastic polymer. Our study\noffers valuable insights into the structural stability of these enzymes\nand the molecular determinants driving PET binding onto their surfaces,\nsheds light on the mechanistic steps that precede the onset of hydrolysis,\nand provides a foundation for future enzyme optimization.