3. Results and discussion:
3.1 Bioinformatics-based determination of Tf C residues crucial for PET binding
Figure 1A shows a Tf C-2HE-(MHET)3 complex. Hydrophobic, electrostatic, hydrogen bonding and van der Waals interactions between Tf C and 2HE-(MHET)3 in this docked structure are illustrated in Figure 1B. Residues observed to participate in interactions with 2HE-(MHET)3 were confirmed using Molecular Dynamics (MD) simulations. The MD simulation interaction diagram (SID) is shown in Figure 1C, plotting fraction/extent of interactions during 75 ns simulations. With PETase (PDB ID: 6ANE) [Fecker et al., 2018], and LCC (PDB ID: 4EB0) [Sulaiman et al., 2012], respectively, Tf C (PDB ID: 4CG1) [Roth et al., 2014] shows RMSD alignment values of ~1.26 Å, and ~1.04 Å. We comparedTf C’s structure with those of PETase, and LCC, in respect of analogous residues (in and around the active site) that make contact with 2HE-(MHET)3 in docking and MD studies, to decide upon mutations of Tf C residues into residues present at structurally-analogous locations in PETase, or LCC, with a view to examining the effect of such mutations upon Tf C’s activity. Notably, this approach is analogous to recent work in which residues in PETase were replaced by residues in Tf C [Fecker et al., 2018]. Our objectives were (i) to obtain mutants/variants superior to Tf C, and (ii) to analyse reductions or improvements in activity, in light of mechanistic and catalytic insights.
The mutants made are listed below. G62A : G62 is located in the PET-binding groove (Figure 1A). Mutation G62A reduces inhibition ofTf C by MHET and increases activity by ~2.0-fold [Wei et al., 2016], ostensibly through disruption of steric clashes preventing interactions (required for release of product after hydrolysis) of residue G59’s backbone nitrogen atom with a carbonyl oxygen in PET. We used G62A as a control, and also as a background (base) mutation for other mutations. L90A, L90F (F125 in LCC, otherwise conserved): L90 is distal to Tf C’s PET binding groove (Figure 1A and Supporting Information Figure S1). L90A shows a 5-fold increase in activity upon cutin, over Tf C [Dong et al., 2020]. Mutations L90A and L90F were made to examine effects on activity. H129W (W132 in PETase [Fecker et al., 2018]): PETase is more catalytically active than Tf C between 30 and 40 °C. To mimic PETase’s catalytic environment around S130 in Tf C, mutation H129W was performed (Supporting Information Figure S2A), in place of H129A, already reported to reduce PET hydrolysis by 80% [Fecker et al., 2018]. W155F (W conserved in all PET hydrolases), H184A, and H184S (S187 in PETase): These mutations were made with a view to create more space in Tf C’s OET/PET-binding cleft towards more efficient PET binding (Supporting Information Figure S2A). H184 was made to allow a nearby tryptophan to wobble between open and closed conformers, as in PETase [Han et al., 2017]. A173C/A210C (C174/C210 in PETase) andA173C/A206C : These mutations were jointly made to create a disulphide bond below Tf C’s catalytic site, mimicking such a bond in PETase [Han et al., 2017], to study its role in catalysis (Supporting Information Figure S2B). ΔV164 : V164 exists in a loop following a beta strand leading to D176 (a member of Tf C’s catalytic triad). This was deleted to bring D176 closer to the other two residues in the triad, to promote better catalysis by minimizing distance between D176 and H208 (Supporting Information Figure S3).F209I (S211 in PETase), F249R , and F249A (not conserved): These residues lie either near or somewhat away fromTf C’s active site. Mutations were made to reduce surface hydrophobicity without compromising catalysis (Supporting Information Figure S1). General : L90, F209 and F249 (Figure 1A), were mutated to either increase hydrophobicity (e.g., L90F) or reduce it (e.g., F209I, F249R). Other residues shown in red (Figures 1B and 1C) were mutated to gain mechanistic insights.