Fig. 2: Computational redesign of the PET hydrolase Bhr-PETase and experimental characterization of the designed variants.

a Design sites of PET hydrolase Bhr-PETase. In the left image, the PET model substrate is shown in a stick model, while the catalytic residue S165 is marked with a ball-and-stick model. In the right image for enlarged view, the catalytic triad residues (S165/H242/D210) are colored in green, and the amino acid type change residues are colored in gray. The hydrogen bonds are shown in dashed lines colored in yellow, around which the numbers indicate the hydrogen bond distances (Å). b, c Catalytic indicators calculated from the high-throughput MD simulations (10 × 1 ns) for the designed variants. The blue triangles represent the computationally designed variants, while the orange squares represent the experimentally evaluated variants. b The abscissa represents the probability of hydrogen bonding between oxyanion hole residue Y95 and the PET model substrate, and the ordinate represents the probability of hydrogen bonding between oxyanion hole residue M166 and the PET model substrate. The hydrogen bonding criteria used for catalytic indicators are: the bond length (donor-acceptor) < 3.5 Å, and the bond angle (donor-H-acceptor) >120°. c The abscissa represents the probability of the nucleophilic attack distance ((S165) OG-C16 (PET)) being less than 3.5 Å, and the ordinate represents the probability of the RMSD of the PET model substrate is less than 2.0 Å. d Catalytic activity and thermal stability evaluation of the designed variants. The ordinate represents the ratio of PET conversion for the variants compared to that of the wild-type. Each variant was tested at a temperature of 68 °C for 12 h, using 0.16% (w/v) of Pc-PET powder and 1 mg_enzyme g_PET⁻¹ of enzyme in 100 mM phosphate buffer (pH 8.0). The T_m values of the variants were measured using the DSF method, in which the samples were heated from 25 °C to 110 °C at a rate of 2 °C min⁻¹. All reactions were carried out in triplicate. e Computed binding geometries of PET model substrate and the wild-type Bhr-PETase based on the crystal structure (PDB ID: 7EOA). The Van der Waals repulsion between two atoms is shown in dashed lines colored in red, around which the numbers indicate the distances (Å). The β6-β7 loop is shown in the purple cartoon model. In Bhr-PETase, the closest distances from the side chain and backbone of residue W190 to PET model substrate are 2.3 Å ((W190) CG-C18 (PET)) and 2.7 Å ((W190) CB-C18 (PET)), respectively. The closest distance of residue F243 to PET model substrate is 2.8 Å ((F243) CE2-O7 (PET)).