On the Role of Temperature in the Depolymerization of PET by FAST‐PETase: An Atomistic Point of View on Possible Active Site Pre‐Organization and Substrate‐Destabilization Effects

Enzyme FAST‐PETase, recently obtained by a machine learning approach, can depolymerize poly(ethylene terephthalate) (PET), a synthetic resin employed in plastics and in clothing fibers. Therefore it represents a promising solution for the recycling of PET‐based materials. In this study, a model of PET was adopted to describe the substrate, and all‐atoms classical molecular dynamics (MD) simulations on apo‐ and substrate‐bound FAST‐PETase were carried out at 30 and 50 °C to provide atomistic details on the binding step of the catalytic cycle. Comparative analysis shed light on the interactions occurring between the FAST‐PETase and 4PET at 50 °C, the optimal working conditions of the enzyme. Pre‐organization of the enzyme active and binding sites has been highlighted, while MD simulations of FAST‐PETase:4PET pointed out the occurrence of solvent‐inaccessible conformations of the substrate promoted by the enzyme. Indeed, neither of these conformations was observed during MD simulations of the substrate alone in solution performed at 30, 50 and 150 °C. The analysis led us to propose that, at 50 °C, the FAST‐PETase is pre‐organized to bind the PET and that the interactions occurring in the binding site can promote a more reactive conformation of PET substrate, thus enhancing the catalytic activity of the enzyme.


Introduction
Since 1941, when poly(ethylene terephthalate) (PET) was patented, its extended and intensive use has caused severe environmental pollution that has only recently begun to be properly tackled.The amount of plastic waste generated annually consists indeed of more than 260 M-tons, an amount that is destined to double by 2030. [1,2]Furthermore, ~52 M tons/year of this waste is incinerated for energy recovery, while ~100 M tons/year is sent to landfills, predominantly in poor communities that will likely bear the burden of its toxic legacy raising global problems. [3]he plastics accumulated and dispersed in the environment are transformed overtime into very small particles, the so-called micro/nanoplastics, that not only contribute significantly to the emissions of greenhouse gas responsible for climate change, [4] but also have harmful, both acute and chronic, effects on humans, such as the occurrence of contact dermatitis.Several studies have shown that microplastics are accumulated in our body [5] causing health effects such as diabetes, obesity and infertility and have also been shown to act as carcinogens. [5,6]ET, as most of other plastic products, is one of the most widely produced thermoplastic polymers for packaging, [7] and its chemical cleavage into its raw materials through environmentally sound solution for its depolymerization is therefore necessary. [8,9] valid proposed solution is the enzymatic degradation [8,[10][11][12][13] throughout a mesophilic bacterium recently isolated that is capable of degrading PET, named Ideonella sakaiensis 201-F6. [14,15]Extensive studies devoted to discovering the catalytic mechanism revealed a synergistic behavior of two enzymes, a PET hydrolase and a mono(2-hydroxyethyl)terephthalate hydrolase (MHETase). [16,17]The enzymes work in tandem because the PETase is responsible for the depolymerization of PET into MHET (mono(2-hydroxyethyl) terephthalate), which in turn is cleaved by MHETase into easily metabolizable products such as the terephthalic acid (TPA) and ethylene glycol (Scheme 1). [18]The structure of both enzymes is characterized by α/β hydrolase domain responsible for catalytic activity. [19]ETase, recognized as the best protein capable of degrading PET, utilizes the catalytic triad Ser160-His237-Asp206 to carry out its activity [20] and, belonging to mesophilic bacterium, works under mild temperature conditions.For this reason, numerous studies have been addressed to improve its efficiency. [21][23] These enzymes, however, are thermophilic, and therefore perform their catalytic activity at high temperatures ( � 70 °C).
An example of such enzymes is represented by LCC, derived from the cutinase class, which is considered as a promising candidate for PET hydrolysis also.It is indeed structurally very similar to PETase and has an identical catalytic triad (Ser165-His242-Asp210). [22,24] However, these enzymes, as mentioned earlier, work at unsuitable temperatures, making them out-ofrange for both the in-situ degradation conditions process and for the cost.In fact, one of the goals for the PET degradation is to set working ambient temperatures (< 30 °C), which would lead to a considerable reduction in costs, thus making the recycling process scalable. [14]iven this, PETase seems to be the ideal candidate to degrade the plastic material but it is highly unstable at these temperatures and can easily lose its activity.][26] The most notable obtained PETase variants are ThermoPETase [24] and DuraPETase, [25] which, although an improvement of both stability and enzyme activity has been obtained, in some states still show lower hydrolytic activity than the wild-type (wtPETase) protein at ambient temperatures (Figure 1). [24,25]A recent study, using machine learning techniques based on 3D convolutional neural network (CNN), identified stable mutations for the PETase enzyme [27] and allowed to focus on one of these variants that has improved hydrolytic activity, who was named FAST-PETase (FAST-: functional, active, stable, and tolerant; Figure 1).This engineered enzyme, based on the ThermoPETase scaffold, [24] contains five mutations compared to the wild-type form (Asn233Lys-Arg224Gln-Ser121Glu-Asp added by the prediction and Asp186His-Arg280Ala based on the scaffold) and shows a wider working temperature range (30-60 °C), with an optimal working temperature of about 50 °C, and a superior catalytic activity in comparison to the wild-type enzyme and to the other already engineered forms.An additional and remarkable aspect of FAST-PETase is the ability to completely degrade a thermally pre-treated water plastic bottle at 50 °C. [27]Beyond the remarkable and promising improvement, the knowledge of working mechanism of FAST-PETase still lacks atomistic details and in-depth analysis starting from the early binding step is required.][30][31][32] In particular, it was pointed out that the plasticity of wtPETase can allow the adaptive accommodation of the substrate in the active site of the enzyme, prior to the catalytic reaction, and that the productive binding is mainly related to a flexible β1-β2 connecting loop. [32]Additional insights on the selection of trans-gauche orientation of PET by the enzyme were further obtained. [31]n this scenario, we decided to carry out a computational investigation applying molecular docking and classical molecular dynamics (MD) techniques on the engineered FAST-PETase, in its apo-and its corresponding complexed forms, considering a tetramer PET model as substrate.In detail, we focused on the structural and dynamic properties of FAST-PETase enzyme, at the lowest temperature of the action range for the system that  is, at 30 °C and at the optimal working temperature of 50 °C, [27] with femtosecond time resolution and at the atomistic level.
MD simulations were used to investigate the effect of different temperatures to FAST-PETase and to the substrate, shedding light on protein-substrate and substrate-substrate interactions that may occur during the binding step of the catalytic mechanism and that we propose as source of the improved FAST-PETase enzymatic activity.At this scope, computer simulations and molecular modeling revealed to be effective in providing deeper information that are not always accessible by applying other techniques.Authors thus believe that the obtained results at molecular level can enhance research in this field, providing important insights into the industrial scalability of these enzymes.

Computational methodologies
Molecular dynamics.The initial coordinates of the FAST-PETase were obtained from the X-ray structure PDB ID: 7SH6. [27]In all the simulations, the ff99SB force field was selected to describe the protein.The studied polymer substrate was modeled considering four monomers (4PET; Scheme 2), as already found in other in silico investigations. [15,29]The substrate parameters were obtained from HF/6-31G* optimizations using the Gaussian16 D.01 package. [33]To extrapolate the parameters and unbound charges, the General Amber Force Field (GAFF) and Restrained Electrostatic Potential methods were adopted, respectively (see the Supporting Information for details). [34,35]MDs simulations of both apo-and substratebound forms of the protein were conducted at the 30 °C (303 K) and 50 °C (323 K).The FAST-PETase was placed in a 62×61×58 Å 3 cubic box containing water molecules as a buffer and with counterions, to set the total charge zero for each considered case.Starting from the 3D models, the systems were initially minimized and relaxed by applying harmonic positional constraints on all atoms (50 kcal mol À 1 Å -2 ) using 5000 steepest descent (SD) steps, followed by 5000 conjugate gradient (CG) steps.In the second minimization step, the entire system was unconstrained and then progressively heated to 30 and 50 °C for 20 ns, using the Langevin thermostat in the NVT ensemble.To produce MDs, 300 ns trajectory were collected, keeping the systems at 1 bar pressure in the NPT ensemble and selecting the Berendsen barostat with a time constant τp = 2.0 ps.In all the simulations, the SHAKE algorithm, and Particle Mesh Ewald (PME) summation method were adopted, with an integration step of 2 fs and a cutoff radius of 10.0 Å, and the water molecules were treated with the TIP3P scheme.
[38][39] The intrinsic conformational behavior of the 4PET in solution (34×30×38 Å 3 TIP3P water box) was further investigated, via three different MDs at 30 °C (303 K), 50 °C (323 K) and 150 °C (425 K).The final production, in each condition, was carried out for 150 ns.In summary, 1.2 μs for apo FAST-PETase, and its complexed form, and 0.45 μs of MDs for 4PET systems were performed.Additional 1.2 μs of MD simulations (3x100 ns replicas for the four systems) were preliminary carried out on the investigated systems, to detect any relevant conformational change.
For comparison purposes with the conformational behavior of FAST-PETase, MD simulations on wtPETase (PDB ID: 5XJH) [40] were also performed at 30 °C and 50 °C (3×100 ns replicas for both apo and 4PET-bound systems), to obtain 1.2 μs of MD simulations.In total, 3.65 μs MD simulations were carried out for the present investigations.
Details about the results and the adopted parameters are described in Supporting Information file.All the simulations were performed adopting the Gromacs package. [41]th the aim of capturing the representative conformation for each system, geometric clustering based on root mean square deviation (RMSD) was performed to identify similar structures sampled during the MD simulation, using the cpptraj module as implemented in AMBER16, choosing a cut-off of 0.7 Å.The binding free energies between the FAST-PETase and the 4PET substrate at different temperatures were calculated by solving the linearized Poisson-Boltzmann equation using the molecular mechanics-generalized Born surface area (MM-GBSA) method, implemented in the Amber16 code. [42]The igb flag value of 5, associated with a salt concentration of 0.1 M, was used.For the calculations, 200 frames of each MD trajectory over the last 100 ns were analyzed.Molecular docking.To investigate possible binding modes and substrate interactions in the vicinity of the catalytic triad of the FAST-PETase protein (Ser160, His237, Asp206), molecular docking simulations were performed using AutoDock version 4.2. [43]Each representative structure was prepared by assigning atom types and adding Gasteiger charges to FAST-PETase protein and 4PET substrate.The docking area was established using AutoGrid.A size of 46×46×46 Å 3 was chosen and the grid was centered on the Cα of Ser160.The Lamarckian genetic algorithm (LGA) was used for the conformational search of the ligand.The docking calculations were performed on the X-ray structure of the protein, with a population size of 150, random initial position and conformation, local search rate of 0.6 and 2,500,000 energy evaluations.The final docked poses were grouped using an RMSD tolerance of 2 Å.

MD simulations of FAST-PETase apoform revealed preorganization of the active site
As largely reported, molecular docking and molecular dynamics simulations can play an important role also for corroborating conclusions validated by means of experimental assays. [17,44,45]In the present study, we performed 300 ns of MD simulation on apoform FAST-PETase at 30 and 50 °C, starting from the crystallographic structure of the enzyme.During the simulation, an equilibrated trend of RMSD has been observed for both temperatures.The analysis of representative geometries arising Scheme 2. The 4PET model adopted in this investigation.from hierarchical clustering procedure indicated that a conformational homogeneity was kept by the system, with respect to the X-ray structure in both cases (Figure S1 in the Supporting Information).However, some differences, in terms of conformational behavior, were observed for FAST-PETase at the selected temperatures.In particular, the analysis of RMSD calculated for backbone atoms of the protein highlighted a slight shifted trend in the case of 50 °C with respect to 30 °C, with values oscillating in proximity of 1.2 and 1 Å (Figure S1), respectively.The RMSD trend calculated for the wtPETase enzyme revealed instead values centered to about 2 Å, at 30 and 50 °C, which can be linked to the higher stability of mutated enzyme (see the Supporting Information file for further details). [27]The obtained RMSD trend for the wtPETase is further in agreement with the recently published results on the Arg103Gly/Ser131Ala mutated enzyme, observed in 500 ns of MD simulation. [32]espite the visual inspection of MDs trajectories did not highlight any relevant variation of FAST-PETase secondary structures, the analysis of root mean square fluctuation plot (RMSF; Figure 2A) revealed some differences.The protein at 30 °C showed a major fluctuation in proximity of loop Glu204-Pro210, caused by a major re-orientation of Asp206 and, consequently of the loop itself.This solvent-exposed residue of the catalytic triad (Ser160-His237-Asp206) tended to re-orient its carboxylate group during the simulation, due to hydrogenbond interactions with water molecules, and some variations of the distances involving the catalytic residues as evinced in Table S1.Such conformational re-organization was indeed observed in the principal component analysis (PCA) of FAST-PETase at 30 °C (Figure 2B), which highlighted how the loop Glu204-Pro210 is the main shifting region of FAST-PETase.It is interesting to point out that from the comparison between the two considered temperatures, several higher peaks were observed for the MDs of apoform protein at 50 °C.In first instance, slightly higher values were registered for the amino acids of the catalytic triad, due to water-bridged hydrogen bonds with surrounding Trp159 and Trp185 amino acids.More interestingly, the regions involved in major fluctuations correspond to the antiparallel β-sheet of Val52-Thr56 and Val106-Asp112 and loops in the segment Ala65-Gly79.In addition, the PCA calculated at 50 °C revealed movements of Gln237-Met262 and Lys233-Asn246 α-helices, with latter peptide sequence corresponding to one of the regions where machine-learning suggested mutation occurred. [27]Such fluctuations generated a network of vicinal grooves in the protein cleft, where the substrate is supposed to bind, and the reaction takes place.The presence of these grooves, extended on the solvent-exposed side of the protein (see shaded areas in Figure 2C), made the FAST-PETase richer of sites to accommodate 4PET substrate and expanded the enzyme surface available for protein-substrate interactions (Figure 2).
A number of these pockets were thus filled by a higher number of water molecules due to the higher kinetic energy generated by the different temperature, as further confirmed by the calculation of solvent-accessible surface area (SASA) at 30 and 50 °C, reported in Figure S1.A consequence of this movement might be represented by the more frequent interactions with the solvent-exposed amino acids that led to the spreader fluctuations of many of the above-mentioned regions of the protein, keeping the loop Glu204-Pro210 in similar orientation for the entire dynamics at 50 °C.
The observed behavior led us to hypothesize that the higher temperature might favor the pre-organization of the active site, which at 50 °C is tailored to bind/react with the substrate.This fact was further confirmed by analyzing MD trajectories of 4PET binding systems, as will be shown in the next paragraph.

FAST-PETase promotes solvent-inaccessible conformation of 4PET that can be relevant for the catalysis
In the following section, the comparative analysis of FAST-PETase:4PET binary complex at 30 and 50 °C is presented.As mentioned in the Methods section, the starting proteinsubstrate complex was chosen from the docking pose having the best binding energy of À 3.4 kcal mol À 1 (Figure S2) and later was subjected to MDs at 30 and 50 °C.This protocol represents an alternative to that already applied in similar systems where, to obtain multiple orientation of the substrate in the active site of both wild-type and double mutant PETases, multiple PET orientations were predicted by induced fit docking (IFD). [26,29]ery interestingly, the FAST-PETase:4PET binary complex showed a different structural behavior as a function of the selected temperature.In accordance with the results obtained for the apoform, RMSD analysis of protein-substrate complex evidenced a shift to higher values in the case of 50 °C (~1.5 vs. ~1.0Å for 30 °C; Figure S3).RMSF plots indicated that the Glu204-Pro210 loop and Val211-Pro217 portion of the adjacent helix significantly fluctuated at 50 °C during the simulation (Figure 3).
The movement of the above-mentioned loop resembles an open-closed conformation with respect to the opposite Pro85-Arg90 loop; this gives birth to the active-site cleft, which is strictly correlated to the ability of the enzyme to accommodate the substrate and which characterizes FAST-PETase protein.Such a cleft's width was approximately 9 Å for both considered temperatures and 0.5 Å larger than in the case of wtPETase [15] and of other cutinases that were previously compared to the wild-type enzyme (Figure 4A and B). [26]This bigger cavity allowed the conformational rearrangement of 4PET substrate in the protein.Indeed, during the simulations, several conformations were observed for the substrate.At 30 °C, the aromatic groups of 4PET tended to establish intramolecular π-π interactions in a range of 4-6 Å, characterizing a wrapped form of the substrate as observed in the radial distribution functions (RDF) calculated for centroids of the 4PET aromatic rings (Figure 4C).Details on each pair are collected in Figure S4.The analysis of simulation at 50 °C showed, instead, a different conformational arrangement of the substrate, which passed from a wrapped form to an unwrapped one (W-shape) after 100 ns of MDs.Such behavior was further confirmed from the occurrence of longer head-to-tail distance of the substrate, which was measured considering the terminal À CH 2 OH groups of the 1st and 4th monomer of the model substrate (Figure 4D).Indeed, using a cut-off head-to-tail distance of 15 Å, the wrapped-like conformations has been observed for the 78 % of the FAST-PETase:4PET simulation at 30 °C, while W-shaped like ones for a total of 28 %.At 50 °C, the wrapped-like and the Wshaped-like conformations were differently populated, as evidenced by the estimated frequencies of 45 and 55 %, respectively.The W-shaped of 4PET conformation here-presented for FAST-PETase resembled the conformations observed in previously published experimental-computational works, devoted to study of wtPETase or some of its mutations with PET models. [26,28,29]It is interesting to note that the occurrence of such conformation can be obtained only by considering an adequate number of model monomers (> 2). [26,28,29]DFT optimizations, performed coupling B3LYP-D3, 6-31G(d,p)basis for all atoms and the SMD implicit model (ɛ = 78), [46][47][48][49][50] of 4PET conformations remarkably revealed the spontaneous trend of the molecule to own a wrapped form in solution.
To evaluate the energy of W-shaped form at DFT level of theory, was therefore necessary to keep fixed the dihedrals of the substrate ester groups.The comparison of relative energies revealed that, in such state, the 4PET lies at 10.2 kcal mol À 1 higher than the optimized wrapped one (Figure S5).This suggested that the enzyme can promote the occurrence of substrate's conformation otherwise non accessible, which might have impact on the hydrolysis of the polymer, as will be highlighted in the next sections of this paragraph.The free energy surfaces (FESs) analysis calculated for the first and second PCAs of 4PET substrate in enzymatic environment supported this hypothesis (Figure S6).In addition, all the MD simulations performed on the wtPETase:4PET system did not show such a W-shaped conformation also, either performing replicas or extending the molecular dynamics to longer simulation time, that is, 300 ns (see the Supporting Information for further details).
When lying in the more opened W-shaped form, 4PET enters in contact with different solvent-exposed residues that compose the binding site of FAST-PETase (Figure 4E and F).Among these, are of particular interest those implicated in the formation of stacking interactions (face to face or point-to-face) that allow the good engagement by the protein counterpart.The most frequent interaction with the Tyr87 has been identified from the calculation of RDFs obtained for the 4PET aromatic rings and the Tyr87 phenol moiety, at 50 °C.The peak resulting at around 4 Å can be reasonable linked to the presence of a frequent π-π stacking occurring between 4PET substrate and the amino acid.
In analogy with the Tyr87, less frequent π-π interactions with the Trp159 and Trp185 were identified from RDF calculations, with highest peaks obtained in proximity of 5 Å.Trp159, in addition, represents a key residue in proximity of the active site owing to X1 of the consensus sequence Gly-X1-Ser-X2-Gly. [15]Interestingly, the RDFs at 30 °C highlighted a remarkably different behavior of π-π interactions occurring between the 4PET and FAST-PETase enzyme.Indeed, for all the three considered pairs, five times lower or 1 Å distance-shifted peaks resulted.These results are a direct consequence of the different conformations observed at the selected temperatures, being the W-shaped conformation at 50 °C more spread on the protein surface than the wrapped form at 30 °C.In addition, the resulting occurrence of π-π interactions with the aromatic amino acids, led to hypothesize that Tyr87, Trp159 and Trp185 support the detection from the solution, and the binding, of 4PET, prior to the catalytic reaction and that, furthermore, the "antenna function" can be favored by the temperature, as analogously discussed in recent review. [28]MMPBSA calculations further revealed a better binding affinity of the enzyme in the W-shaped 4PET (ΔΔE = 15 kcal mol À 1 ; Figure S7) in comparison with that in spiral-shaped one, a trend that can be further related to the occurrence of a more frequent protein-substrate interactions.
For the active site, the catalytic triad behavior was monitored in terms of relevant distances between amino acids, like Ser160-His237 and His237-Asp206 (Figure S8).It is possible to note that no remarkable differences emerged at 30 and 50 °C.However, in the case of the MD simulation at 50 °C, the distance of OH moiety of Ser160 from Nδ of His237 presented a larger span of values than the corresponding one at 30 °C.The obtained distances are anyway coherent with a suitable orientation of the amino acid residues for the occurrence the catalysis since, as recently suggested, the activation of Ser160 borne by the His237 can be mediated by water molecules bridging the residues. [9,30]From the RDF analysis performed at both temperatures and obtained as a function of the distance between the water oxygen and the side chain of Ser160, it was possible to detect the different hydration shells starting from 2.5 Å that are characteristic of extensive network of H-bonds with the closer water molecules.At 50 °C, Ser160 interact also with Trp185 throug H-bond formation between its alcoholic function and the À NHÀ of the indole group (Figure S10).
This behavior favors a different orientation of the aromatic amino acid, not observed at 30 °C, and increases the frequency of π-π stacking with 4PET, thus highlighting a possible correlation to the more pronounced plasticity of the enzyme at 50 °C (Figure 3B) evidenced at experimental level. [27]Upon the occurrence of this interaction, the substrate and its eight carbonyl groups that can undergo to the catalytic mechanism in detail, resulted averagely at closer distance from the À OH group of Ser160 and from the À NHÀ group of Met161 backbone.According to the proposed catalytic reaction, the former residue is the nucleophilic agent, while the latter is deputed to generate the oxyanion hole for stabilizing the transition states and intermediates, together with the cooperative action of À NHÀ group of Tyr87. [27,28]Therefore, the OH Ser160 À C=O 4PET and NH Met161 À O=C 4PET were measured during the simulations at 30 and 50 °C, taking into account each target carbonyl group of 4PET (Figures S11 and S12).The focus on Met161 was supported by the analysis of RDFs calculated between every C=O 4PET and the backbone amino groups of Tyr87 and Met161 that indicated a more frequent interaction with the latter residue (Figure S13).A cut-off of 5 Å for both selected distances was later adopted to identify possible catalytically productive conformations of 4PET.Overall, it turned out that, at 50 °C, the relative abundances of good orientations of the substrate are observed for both OH Ser160 À C=O 4PET and NH Met161 À O=C 4PET distances for many moieties that are involved in the catalytic mechanism (Table S2).More interestingly, at this temperature, the productive distances were observed for both core-and terminal monomers, examining the OH Ser160 À C=O 4PET distances (Figures S11 and S12), as consequence of the more extended W-shape form.Such more frequent proximity of 4PET carbonyl groups to the catalytic residues suggested that FAST-PETase enzyme, in these working conditions, can increase the chances to catch the substrate in a productive way for the catalysis from the early step of the catalytic mechanism, where efficient stabilization of transient intermediates is required.In absence of further indications about the preference for core-or terminal-monomers of FAST-PETase, such results can be nicely considered representative of, and can thus linked with, the enhanced catalytic activity of FAST-PETase, which was experimentally observed and measured in terms of yielded TPA and MHET monomers. [27]Furthermore, the W-shaped substrate resulted in hydrogen-bond interaction with the solvent exposed guanidine group of the Arg90, as evidenced by the RDF obtained for the center of mass of amino acid side chain and the terminal ester group of 4PET (intense peak at 2.8 Å at 50 °C, Figure 5A).On the contrary, the highest value at 30 °C was found in proximity of 10 Å (Figure 5A).A salt bridge occurring between Lys233 and Glu204 was observed at 50 °C as shown in Figure 5B.The charged side chains of the amino acids were faced at 3 Å for the 30 % of molecular dynamics at 50 °C, while the highest percentage of 30 % was obtained in proximity of 8 Å at 30 °C.
The keeping of this interaction favored the W-shaped form at higher temperature, thus highlighting the effectiveness of machine-learning based Lys233Asn mutation coupled to the selection of 50 °C as working temperature.Indeed, during the MD simulations on the wtPETase.4PETcomplex, the opened conformation did not occur also (see the Supporting Information for details).

W-shaped conformation of 4PET was not observed in solution
In accordance with the above-discussed results, it was pointed out that an increasing temperature can favor FAST-PETase:4PET interactions, promoting accessibility to high-energy conformations of the substrate.To further verify this hypothesis, three different molecular dynamics simulations of 150 ns were performed on the 4PET in solution, at increasing values of temperature (30, 50 and 150 °C).[53][54] However, the choice of the temperatures higher than 50 °C allowed to evaluate the thermic stress on the conformation in absence of protein surroundings to the selected substrate model.
Over the selected timeframe and under the temperature conditions, 4PET never showed a conformation resembling Wshaped form, as can be evinced by FESs calculated for PCA1 and PCA2 in Figure S14.At 30 and 50 °C there were three main free-energy wells/basins in the global free energy minimum region, indicating mainly three stable wrapped states characterized by π-π staking interactions (A, B, and C; Figures S14 and  S15) over a range of ~4 kcal mol À 1 .The mutual local structural organization within the 4PET was analyzed in terms of RDFs for each temperature considering all the four rings and their related distributions (Figure S16).It was interesting to note that, at an increasing temperature, a decreasing height of the peaks of about 4 Å can be observed, thus emphasizing that the temperature can favor a conformational distribution shifted towards the extended conformation.However, the analysis of FES at 150 °C did not still present any W-shaped form of the substrate (Figure S17), in analogy to those conformations observed for wtPETase and for FAST-PETase at 30 °C also (see results from Figures S18-S21).
57] Despite the use of a representative model, it is worth nothing that the here-described simulations reasonably reproduced, at atomistic level, the conformational behavior that might be representative of what occurs in PET-based material.Thus, the insights obtained from these simulations can represent indirect evidence of the role played by FAST-PETase enzyme, which, with its engineered protein architecture, owns a binding site that promote the access to more reactive conformations, which would not spontaneously occur for the polymer itself.This aspect is further in accordance with previous in silico works on other mutants of wtPETase. [28]

Conclusions
In this study, MD simulations were carried out to provide indepth details of the atomistic interactions of PET and FAST-PETase protein.This enzyme represents a machine-learningbased improvement on PETase from Ideonella sakaiensis and showed enhanced hydrolytic activity of PET.It has therefore been recently proposed as good agent for the disposal of plastic-based wastes.
A model of the apoform FAST-PETase was built up, starting from a recently deposited crystal structure of the enzyme to study its conformational behavior at 30 and 50 °C through MD simulations, with the latter condition being the optimal working one.A tetramer model of PET (4PET) was initially docked to the FAST-PETase and later subjected to MD simulations at both temperatures.
It has been pointed out that the protein's response is dependent on the selected temperature for the simulation.In detail, a more widely spread and higher number of fluctuations was detected for apoform FAST-PETase at 50 °C in proximity to protein's active-site cleft where the binding of the 4PET is hypothesized to occur.The fluctuations generated many grooves on the solvent-exposed side of the protein that could be relevant to a more favored binding of the substrate, thus highlighting a pre-organization of the active site at 50 °C.
From analysis of the MD simulations of the FAST-PETase:4-PET complex it turned out the enhanced plasticity in the FAST-PETase at 50 °C led to an improvement in the lipophilicity of the active site that stabilizes the aromatic moieties of the substrate in the right conformation for catalysis, with respect to 30 °C.This is related to the more efficient orientation of aromatic residues such as Trp185 and Tyr87 that anchor the terephthalic rings of 4PET to establish hydrophobic and π-stacked interactions.
The presence of these protein-substrate interactions made possible the occurrence of a W-shaped conformation of 4PET at 50 °C that is characterized by a broadly extended conformation of the substrate over the surface of the binding site of the protein.Such a conformation was not observed in the case of wtPETase enzyme.In contrast to the wrapped one, mainly observed at 30 °C, 4PET better approaches the catalytically important residues, like the nucleophile Ser160 and the oxyanion hole backbone of Met161.This aspect can therefore be linked to the enhanced enzymatic activity observed at 50 °C.The W-shaped conformation was better accommodated in the cleft for the creation of a salt bridge between the mutated Lys233 and Asp204, which was further stabilized by an interaction with Arg90.DFT calculations on the wrapped and W-shaped conformations of the 4PET model proposed the former one as a higher and inaccessible energy conformation of 4PET in solution (lying at ~12 kcal mol -1 above the wrappedshape).Interestingly, MD simulations of 4PET in solution, performed at 30, 50 and 150 °C, further confirmed this behavior as the W-shaped conformation was not observed.This finding further validates the FAST-PETase role at 50 °C to favor the observation of W-shaped conformation.This led us to propose that the FAST-PETase environment at 50 °C can dictate higher-energy conformations of the substrate promoting catalysis in turn through destabilization of the substrate.
In summary, the in silico results agree with the experimental outcomes and showed that the average vicinity of the important FAST-PETase catalytic residues to each monomer of the 4PET substrate and the conformational behavior detected for mutated residues can have an impact on the production of a greater number of monomers at 50 °C.This can therefore be related to an improved catalytic performance of the enzyme.We hope that the atomistic insights provided in this work will stimulate further theoretical and experimental investigations, devoted to making an important contribution to the field of the green and sustainable economy and to optimizing these processes at ambient temperatures.

Scheme 1 .
Scheme 1.The products of PET degradation catalyzed by PETase.

Figure 1 .
Figure 1.The PET hydrolase enzymes.Mutations are indicated in pink, green and blue for DuraPETase, ThermoPETase and FAST-PETase, respectively.

Figure 2 .
Figure 2. A) RMSF plot and B) PCA visualization obtained for apoform FAST-PETase molecular dynamics simulation at 30 and 50 °C.C) Surfaces of representative clustered geometries obtained for apoform FAST-PETase from molecular dynamics simulations at 30 and 50 °C.Red and blue areas are localized in the presence of O and N atoms, respectively, while gray and white indicate C and H, respectively.

Figure 3 .
Figure 3. A) RMSF plot and B) PCA (top and bottom) visualization obtained for FAST-PETase:4PET molecular dynamics simulation at 30 and 50 °C.

Figure 4 .
Figure 4. Focus on the enzymatic cleft in the presence of 4PET substrate, obtained from the representative clustered geometry of FAST-PETase-4PET simulations at A) 30 and B) 50 °C.C) RDF calculated for the center of masses of aromatic ring-ring pairs of 4PET.D) frequency of head-to-tail distance and E), F) RDFs calculated for centers of masses of aromatic ring-ring pairs of 4PET and Tyr87, Trp159 and Trp185 obtained for enzyme-substrate complexes at the two selected temperatures.

Figure 5 .
Figure 5. A) Frequency of distances measured for the salt-bridge between Lys233 and Glu204 residues and B) and RDF calculated for center of mass of the guanidium side chain of Arg90 and terminal OH group of 4PET obtained for FAST-PETase-4PET complex at 30 (yellow) and 50 °C (orange).