Reaction Mechanism of MHETase, a PET Degrading Enzyme

Abstract

In 2016, one of the two enzymes involved in the polyethylene terephthalate (PET) degradation pathway of Ideonella sakaiensis 201-F6, MHETase, was found to exhibit a strong ability to degrade the PET monomer mono-(2-hydroxyethyl)­terephthalate (MHET) at room temperature, converting it back into the precursors used in PET production. MHETase engineering to improve efficiency is an active field that suffers from an incomplete characterization of its reaction mechanism. In this paper, we analyze the reaction mechanism of MHETase using umbrella sampling molecular dynamics simulations at the B3LYP/MM level of theory. The combination of a high theoretical level and extensive sampling generated a very robust computational prediction. We found that MHETase catalyzed the conversion of MHET in two steps, with a rate-limiting step activation barrier of ΔG ⧧ = 19.35 ± 0.15 kcal·mol–1 (from the weighted-histogram analysis). Our calculations are in line with the hypothesis that a transient tetrahedral intermediate mediates the reaction mechanism in each step, which is quite common in the serine hydrolase class. The energy of the first tetrahedral intermediate was similar to that of the reactant state, while the tetrahedral intermediate of the deacylation step was observed to lie closer to the rate-limiting transition state. Nevertheless, both determined tetrahedral states were found to be transient, with activation barriers close to ∼2.0 kcal·mol–1 relative to the product state of the acylation and deacylation steps, corresponding to a half-life of about 3 ps at 303.15 K.

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

“…In a previous work of Knott et al for MHETase, where they carried out QM/MM simulations at the SCC-DFTB:MM level, the authors suggested that the reaction occurred in two steps without the formation of stable tetrahedral intermediates. An alternative approach by Pinto et al , also for MHETase, at the B3LYP/GPW:MM level, indicated that the reaction mechanism obtained is analogous to that of canonical serine hydrolases, where the acylation and deacylation steps occur via a metastable tetrahedral intermediate. Here, we used a robust QM/MM umbrella sampling method, at the density functional theory level (PBE) with a larger QM region (146 atoms), and identified all its intermediates and transition states with high accuracy.…”

Chemically recyclable polymer materials: polymerization and depolymerization cycles

“…To better summarize these elegant examples, the review is categorized into seven parts based on the monomers including threemembered ring monomers, four-membered ring monomers, five-membered ring monomers, six-membered ring monomers, seven-membered ring monomers, eight-membered ring monomers, and linear monomers, while some important materials such as PET and BPA-PC were not introduced in this review because they have been reviewed in great detail by others. [29][30][31][32][33][34] In the following, examples for achieving the polymerization-depolymerization cycle of the polymer are presented. By doing so, we aim to highlight the current and future studies in this rapidly growing and promising area.…”

Conformational Selection of a Tryptophan Side Chain Drives the Generalized Increase in Activity of PET Hydrolases Through a Ser/Ile Double Mutation

“…Consistently with this, the S214H mutant of IsPETase shows decreased activity when compared to the wild-type enzyme. 13 We note that there have been multiple elegant computational studies that have helped reveal the mechanisms of PET-degrading enzymes, [15][16][17][18] more global conformational changes involved in catalysis, 19,20 and contributed to associated engineering effort. [21][22][23][24] However, corresponding studies of the conformational dynamics of this catalytically important tryptophan remain limited.…”