QM/MM Study of the Enzymatic Biodegradation Mechanism of Polyethylene Terephthalate

Abstract

The environmental problems derived from the generalized plastic consumption and disposal could find a friendly solution in enzymatic biodegradation. Recently, two hydrolases from Ideonella sakaiensis 201-F6 and the metagenome-derived leaf-branch compost cutinase (LCC), more specially the improved ICCG variant, have revealed degradation activity toward poly ethylene terephthalate (PET). In the present study, the reaction mechanism of this polymer breakage is studied at an atomic level by multiscale QM/MM molecular dynamics simulations, using semiempirical and DFT Hamiltonians to describe the QM region. The obtained free energy surfaces confirmed a characteristic four-step path for both systems, with activation energies in agreement with the experimental observations. Structural analysis of the evolution of the active site along the reaction progress and the study of electrostatic effects generated by the proteins reveal the similarity in the behavior of the active site of these two enzymes. The origin of the apparent better performance of the LCC-ICCG protein over PETase must be due to its capabilities of working at higher temperature and its intrinsic relationship with the crystallinity grade of the polymer. Our results may be useful for the development of more efficient enzymes in the biodegradation of PET.

IsPETase- and IsMHETase-Catalyzed Cascade Degradation Mechanism toward Polyethylene Terephthalate

“…The first step catalyzed by IsPETase, Ser-His-Asp-initiated nucleophilic attack, is determined to be a concerted step rather than stepwise steps as indicated in Figure 3, which is in agreement with a recent theoretical study. 40 A more detailed discussion is provided in the Supporting Information. Typically, this step can be described as follows: His237 abstracts a proton (0.47 e) from Ser160 and the partially charged oxygen (−0.71 e) attacks the carbonyl carbon of the ester bond, forming a negatively charged (−0.57 e) tetrahedron intermediate (IM3).…”

“…21 As a result, thermophilic PET hydrolases are considered more practical for industrial application than mesophilic enzymes and more attentions should be paid to thermostable enzymes in the future. 15,21,[23][24][25][26]40 On the other hand, IsPETase is one of the most effective enzymes that can degrade amorphous PET at ambient temperature and is thus still worth studying. 14,[29][30][31]41,42 Rational protein engineering with comprehensive mechanism understanding is an effective strategy for engineering enzymes.…”

“…14,[29][30][31]41,43 Recently, Boneta et al compared the depolymerization mechanism of PET catalyzed by IsPETase and leaf-branch compost cutinase (LCC-ICCG) with theoretical approaches, where they showed that the depolymerization mechanism of two enzymes is similar. 40 Here, with the high level (DFT) quantum mechanics/ molecular mechanics method (QM/MM), the detailed cascade degradation process of IsPETase and IsMHETase was fully explored in a step-by-step manner. Recently similar strategies have been successfully and extensively applied in exploring enzymatic mechanisms.…”

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes

“…This enzyme catalyses the hydrolysis of the PET polymer with the formation of mono-(2-hydroxyethyl)terephthalic acid (MHET) and, to a lesser extent, TPA and bis(2-hydroxyethyl) terephthalate (BHET); the latter is further hydrolysed to MHET and EG, Figure 1 E [ 10 ]. IsPETase was selected because of the availability of a considerable information on its structure–function relationships and enzymatic mechanism [ 13 , 14 , 15 ]. Although IsPETase shows a lower thermal stability in comparison to homologues cutinases active on PET [ 6 ], its higher esterase activity at mild temperatures and its promiscuous specificity on other emerging polyesters (e.g., on polyethylene-2,5-furandicarboxylate) [ 16 ] render this enzyme an interesting potential candidate for several biotechnological applications [ 17 ].…”

Assessment of IsPETase-Assisted Depolymerization of Terephthalate Aromatic Polyesters and the Effect of the Thioredoxin Fusion Domain

“…A key enzyme, IsPETase (EC 3.1.1.101), was identified as being responsible for PET depolymerization, which showed more activity than others when compared at mild temperatures (27-40 • C) [19,25,26]. Several IsPETase structural studies were published [17,[27][28][29], which set the basis for mechanistic studies [30,31] as well as for the development of a series of mutants with increased activity [32] or thermostability [33][34][35][36][37]. Both thermostable and mesostable PETactive enzymes constitute a valuable toolbox for diverse applications.…”