Reaction Mechanism of the PET Degrading Enzyme PETase Studied with DFT/MM Molecular Dynamics Simulations

Abstract

Polyethylene terephthalate (PET) has been widely used to make disposable bottles, among others, leading to massive PET waste accumulation in the environment. The discovery of the Ideonella sakaiensis PETase and MHETase enzymes, which hydrolyze PET into its constituting monomers, opened the possibility of a promising route for PET biorecycling. We describe an atomistic and thermodynamic interpretation of the catalytic reaction mechanism of PETase using umbrella sampling simulations at the robust PBE/MM MD level with a large QM region. The reaction mechanism takes place in two stages, acylation and deacylation, each of which occurs through a single, associative, concerted and asynchronous step. Acylation consists of proton transfer from Ser131 to His208, concerted with a nucleophilic attack by Ser131 on the substrate, leading to a tetrahedral transition state, which subsequently results in the release of MHET after the breaking of the ester bond. Deacylation is driven by deprotonation of an active site water molecule by His208, with the resulting hydroxide attacking the acylated Ser131 intermediate and breaking its bond to the substrate. Subsequently, His208 transfers the water proton to Ser131, with ensuant formation of MHET and enzyme regeneration. The rate-limiting acylation has a free energy barrier of 20.0 kcal·mol–1, consistent with the range of experimental values of 18.0–18.7 kcal·mol–1. Finally, we identify residues whose mutation should increase the enzyme turnover. Specifically, mutation of Asp83, Asp89, and Asp157 by nonpositive residues is expected to decrease the barrier of the rate-limiting step. This work led to the understanding of the catalytic mechanism of PETase and opened the way for additional rational enzyme engineering.

In Silico Identification of Potential Sites for a Plastic-Degrading Enzyme by a Reverse Screening through the Protein Sequence Space and Molecular Dynamics Simulations

“…However, due to difficulties in co-crystallization and the low solubility of the PET polymer, the PETase structure in complex with PET is still unavailable [ 23 , 25 ]. As a result, only computational approaches, such as molecular docking and molecular dynamics (MD) (i.e., quantum mechanics/molecular mechanics (QM/MM) and a density functional theory (DFT)-based QM/MM) have been employed to investigate their binding mode and molecular interactions [ 22 , 23 , 26 , 28 , 29 , 30 ]. Although the proposed PETase catalytic mechanisms from these studies are similar in that PET depolymerization occurs at the active site of PETase (S160, H237, and A206) with the help of other hydrophobic residues around the binding cleft that brings the carbonyl carbon atom of PET into close contact for nucleophilic attack from S160, the reaction mechanism of Ser–His–Asp-initiated nucleophilic attack is still inconclusive.…”

Cation–π and hydrophobic interaction controlled PET recognition in double mutated cutinase – identification of a novel binding subsite for better catalytic activity

“…From theoretical mechanistic studies on the enzymatic PET hydrolysis, [78][79][80][81][82] it was proposed that the hydroxyl group of Ser130 of the catalytic triad initiates the attack to the carbonyl carbon of the ester bond, followed by the stabilization of the negative charge by the two back bone nitrogen atoms of Tyr60 and Met131 of the oxyanion hole. 83 Thus, in order to identify the best binding poses of PET with WT and DM TfCut2 in terms of catalytic activity, it is very important to analyse the proximity of the carbonyl carbon-atom of PET to Ser130 that lie in the subsite S1.…”

“…8e) in the neutral state, which resembles the state prior to proton abstraction by His208 in the catalytic cycle. 78,80 The aromatic residue Trp155, which is responsible for anchoring the phenyl ring of PET to S1, shows PET C-H/p w155 (À8.1 kcal mol À1 , Fig. 8f) interaction and weak T-stacking interaction with PET (À5.0 kcal mol À1 , Fig.…”

The critical role of Asp206 stabilizing residues on the catalytic mechanism of the Ideonella sakaiensis PETase

“…18,19,[37][38][39] However, some aspects have remained unexplained or subject to debate at the atomic level, 18,19,40 particularly concerning the number of steps involved in the mechanism and the active role of Asp206 residue. With respect to the number of steps, the computational studies of Boneta et al 41 and Feng et al 42 have shown that the reaction occurred in four sequential steps whereas Jerves et al 43 reported a two-steps mechanism. Regarding the role of Asp206, its basic character was not observed in all studies, since some studies have reported the proton transfer from His237 to Asp206 (ref.…”

“…42) and other have proposed that the proton is not transferred and the Asp206 residue only stabilized the positively charged His237 formed during the reaction. 41,43 A detailed QM/MM atomistic description of the catalytic mechanism of this enzyme, dissecting the role played by the different active-site amino acid residues, particularly the Asp206, and by the electronic distribution and charge effects at the transition state would provide valuable insights on bioengineering strategies for increasing their enzymatic efficiency for plastic degradation through rational enzyme mutagenesis.…”