To directly enhance the catalytic activity of the 05PET enzyme, we first utilized structural homology and molecular docking to locate its active pocket, then selected hotspot amino acids, performed mutations, and finally conducted wet experimental validation.

First, we performed molecular docking between the 05PET enzyme and the trimeric long-chain PET molecule using AutoDock Vina^[1]. Unexpectedly, among multiple docking conformations, we did not identify fixed and groove-like binding sites.

Therefore, by comparing with the structure of LCC^[2], we excluded some regions of non-active pockets and redock.

As shown in the figure, the left panel depicts the predicted structure of 05PET (generated by AlphaFold 3), while the right panel illustrates the crystal structure of LCC. The two structures are similar: the β-sheets are situated among multiple α-helices, and the central β-sheets all orient toward the same direction, which point to the notch-shaped enzyme active pocket located on the left part of the figure.

Setting off from this, we defined more reliable docking regions, re-performed molecular docking, and gained the following enzyme pocket interaction results.

Docking results from AutoDock Vina showed that PHE215, HIS214, SER134, ILE186, THR66, GLY65, THR67, TYR73, and ASP184 are amino acid residues that closely interact with the PET substrate, with SER134-HIS214-ASP184 forming its catalytic triad. Considering the active pocket in a spatial context, the amino acids of the active pocket include G54, T55, T56, A57, T62, S124, F145, Q146, Y148, L162, D174, I176, H204, F205, V208, and G209, as shown in the second figure above. Since there are additional sites capable of docking with the PET molecule at exterior region out of active pocket, the zone was appropriately expanded, yielding the first figure above, which was chosen as the structure for modification.

Inspired by evolution, after selecting the sequence sites corresponding to the spatial region, we used the 05PET enzyme as the basis and designed mutations based on the sequences of other PET enzymes, ultimately determining the following single-point mutation schemes:

G55A T56A T56Y T56F T57A T58G T62S T62A Y63I Y63L Y63A H123A H123W S124A Q125M Q125A Y148A Y148W I150A I150T I150L I150S G151A G152A D174A I186A I186V L199A L199I S200N S200A S200D S200R G201A G201N A202G S203A S203T H204A F205A F205L F205S E206A E206C E206V V208N V208A G209A G209I G209T G209S S210A S210N S210G S210P A211N A211D G212A G212T G212K G212S D213A D213I D213N D213T D234A D242A D246A D248A D248E