Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange

Abstract

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.

Keywords: PET hydrolases; mesophilic enzymes; plastics; polyethylene terephthalate; protein engineering.

FIGURE 1

Crystal structure and sequence of Mors1. (a) Cartoon representation of the three‐dimensional structure of chain B of Mors1, with the catalytic residues and three disulfide bonds (DB) in green sticks. The insets correspond to a close‐up of the three DBs, with the black mesh corresponding to the electronic density of each DB. (b) Sequence and secondary structure topology of Mors1. The red boxes indicate the location of the catalytic residues S189, D234, and H264, and the green numbers indicate the cysteines that participate in the three DBs. (c) Partial Multiple sequence alignment derived from the structural superposition of the active sites of Mors1, IsPETase (PDB 6ANE), and LCC (PDB 4EB0).

FIGURE 2

(a) Ribbon representation of a three‐dimensional alignment of Mors1 (navy blue) with homologs LCC (gray) and IsPETase (cyan). The catalytic triad (S189‐H264‐D234) and the non‐conserved residues within binding subsites 1 and 2 are shown. The inset shows a displaced loop (between β‐strands β6 and β7), potentially caused by an electrostatic interaction between D153 and K217. (b) Stick representation of the different conformations observed for Y121 in chains A and B (Y121_A1 in green and Y121_B in cyan, respectively). In yellow, the position that Y121 would adopt in the B subunit if perfect twofold symmetry were retained (Y121_A*) is shown. This position was generated by applying a 180° rotation to Y121_A, giving rise to a severe steric clash.

FIGURE 3

Structure, sequence identity, and length of the extended loop in Mors1 and in other PETases. Cartoon representations depict the experimental structures of Mors1 (a, this work), IsPETase (b, PDB 6ANE), and LCC (c, PDB 4 EB0), and the predicted structure of the chimeric variants of Mors1 (CM and CM^A266C, d) in which the β8‐α6 loop and extended loop were substituted by the equivalent regions from LCC. The disulfide bridge potentially formed in CM^A266C is shown in semi‐transparent sticks. In all cases, the protein is shown in cartoon representation with the β8‐α6 loop and extended loop in yellow, and the catalytic residues and the active site disulfide bond in green sticks. A multiple sequence alignment is shown in (e), showcasing the variations in sequence between these regions for Mors1, IsPETase, and LCC, and the chimeric Mors1 variants CM and CM^A266C. Amino acids are colored by their chemistry: CNQST, green; AGILPMV, orange; RHK, blue; DE, red; FWY, yellow. Numbering corresponds to Mors1.

FIGURE 4

Characterization of the polyesterase activity of loop exchanged variants of Mors1. (a) Enzymatic activity of CM and CM^A266C against a PCL nanoparticle suspension, measured as a decrease in turbidity (AUs/min) at 25°C. (b) Optimal temperature for PET degradation of CM^A266C measured as weight loss of PET films after a reaction time of 24 h. (c) Weight loss of PET films (pink) and HPLC quantification of TPA (dark green) and MHET (light green) released by Mors1 and CM^A266C after a reaction time of 24 h at 25°C and 45°C. WT Mors1 was inactive against PET at 45°C. Mean values ± standard deviation for n = 3 experiments are shown.

FIGURE 5

Local structural flexibility of Mors1 and CM^A266C ascertained by MD simulations. (a) RMSF from 3 × 500 ns MD simulations of Mors1 at 25°C (cyan) and 45°C (blue) and CM^A266C at 45°C (pink), totaling 1.5 μs of simulation per enzyme. The lines represent the average values and the shaded contour around them corresponds to the standard deviation across the triplicates. (b) Cartoon representation of Mors1, with the regions that exhibit increases in RMSF in CM^A266C shown in yellow.