The critical role of Asp206 stabilizing residues on the catalytic mechanism of the <i>Ideonella sakaiensis</i> PETase

Magalhães, Rita P.; Fernandes, Henrique S.; Sousa, Sérgio F.

doi:10.1039/d1cy02271g

Cited by 7 publications

(13 citation statements)

References 68 publications

(111 reference statements)

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“…For PETases to be applied industrially to aid in the depolymerization and recycling of PET, they typically need to be engineered to increase their kinetic and thermal stability, as well as their catalytic efficiency. − These approaches have ranged from random mutagenesis to computational design, machine learning, and rational design. Most mutated residues are targeted to modify the substrate binding site, the wobbling tryptophan residue and those in the vicinity of the disulfide bond in the active site. ,,, As a result, numerous protein engineering efforts have succeeded in developing more stable and active variants, including DuraPETase, ThermoPETase, HotPETase, and FAST-PETase. − Recently, Lu et al demonstrated that FAST-PETase could almost completely degrade untreated, postconsumer PET samples within 1 week and recover the monomers .…”

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confidence: 99%

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity

et al. 2022

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The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

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confidence: 99%

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity

et al. 2022

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“…The reaction mechanism matches that from previous works, suggesting a two-step acylation with the tetrahedral intermediate state a shallow minimum on the free energy surface. 15,17,[22][23][24][25]30,32 Firstly, the catalytic histidine abstracts a proton from the catalytic serine, resulting in TS1. Then, the serine attacks the ester carbon, yielding the tetrahedral intermediate with its oxyanion stabilised by the oxyanion hole.…”

Section: Pet Ring Orientation Change Correlates With Acylationmentioning

confidence: 99%

“…1). [15][16][17][23][24][25][26] Figure 1: Mechanism of PET-degradation as proposed by Boneta et al 15 The mechanism can be divided into two steps: First, the acylation of the Michaelis complex (MC) yielding the acyl-enzyme intermediate (AE) and second, the hydrolysis yielding the product complex (PC), both incorporating a tetrahedral intermediate (TI).…”

Section: Introductionmentioning

confidence: 99%

“…Mutation experiments confirm the importance of isoleucine, with alanine substitutions resulting in activity loss. 23 Such insights are crucial for evaluating mutation sites, particularly in enzyme redesign aiming to lower the free activation energy by stabilising transition states, beyond substrate binding. 33,34 Another interesting residue is a conserved tryptophan, the 'wobbling' tryptophan, which exhibits three different conformations in the mesophilic Ideonella sakaiensis PETase (IsPETase), while maintaining a single conformation in other, mostly thermophilic polyester hydrolases.…”

Section: Introductionmentioning

confidence: 99%

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Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations

Jäckering,

van der Kamp,

Strodel

et al. 2024

Preprint

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Plastic-degrading enzymes, particularly poly(ethylene terephthalate) (PET) hydrolases, have garnered significant attention in recent years as potential eco-friendly solutions for recycling plastic waste. However, understanding of their PET-degrading activity and influencing factors remains incomplete, impeding the development of uniform approaches for enhancing PET hydrolases for industrial applications. A key aspect of PET hydrolase engineering is optimizing the PET-hydrolysis reaction by lowering the associated free energy barrier. However, inconsistent findings have complicated these efforts. Therefore, our goal is to elucidate various aspects of enzymatic PET degradation by means of quantum mechanics / molecular mechanics (QM/MM) reaction simulations and analysis, focusing on the initial reaction step, acylation, in two thermophilic PET hydrolases: LCC and PES-H1, along with their highly active variants, LCC ICCG and PES-H1 FY. Our findings highlight the impact of semi-empirical QM methods on proton transfer energies, affecting the distinction between a two-step reaction involving a metastable tetrahedral intermediate and a one-step reaction. Moreover, we uncovered a concerted conformational change involving the orientation of the PET benzene ring, altering its interaction with the side-chain of the "wobbling" tryptophan from T-stacking to parallel π-π interactions, a phenomenon overlooked in prior research. Our study thus enhances the understanding of the acylation mechanism of PET hydrolases, in particular by characterizing it for the first time for the promising PES-H1 FY using QM/MM simulations. It also provides insights into selecting a suitable QM method and a reaction coordinate, valuable for future studies on PET degradation processes.

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“…We note that there have been multiple elegant computational studies that have helped reveal the mechanisms of PET-degrading enzymes, − more global conformational changes involved in catalysis, , and contributed to associated engineering effort. − However, corresponding studies of the conformational dynamics of this catalytically important tryptophan remain limited. Therefore, to explore the importance of the S214/I218 substitution for PET hydrolytic activity, doubly substituted variants of the strictly conserved histidine and phenylalanine residues in several PET hydrolases, including the mesophilic Bur PL from a Burkholderiales bacterium (H344/F348), and the thermophilic Tf Cut from Thermobifida fusca (H224/F228) and LCC (H218/F222), were generated based on the S214/I218 residues unique to Is PETase.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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Poly(ethylene terephthalate) (PET) is the most common polyester plastic in the packaging industry and a major source of environmental pollution due to its single use. Several enzymes, termed PET hydrolases, have been found to hydrolyze this polymer at different temperatures, with the enzyme from Ideonella sakaiensis (IsPETase) having optimal catalytic activity at 30–35 °C. Crystal structures of IsPETase have revealed that the side chain of a conserved tryptophan residue within an active site loop (W185) shifts between three conformations to enable substrate binding and product release. This is facilitated by two residues unique to IsPETase, S214 and I218. When these residues are inserted into other PET hydrolases in place of the otherwise strictly conserved histidine and phenylalanine residues found at their respective positions, they enhance activity and decrease T opt. Herein, we combine molecular dynamics and well-tempered metadynamics simulations to investigate dynamic changes of the S214/I218 and H214/F218 variants of IsPETase, as well as three other mesophilic and thermophilic PET hydrolases, at their respective temperature and pH optima. Our simulations show that the S214/I218 insertion both increases the flexibility of active site loop regions harboring key catalytic residues and the conserved tryptophan and expands the conformational plasticity of this tryptophan side chain, enabling the conformational transitions that allow for substrate binding and product release in IsPETase. The observed catalytic enhancement caused by this substitution in other PET hydrolases appears to be due to conformational selection, by capturing the conformational ensemble observed in IsPETase.

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

Abstract: Plastic accumulation is one of the main environmental issues of our time. In 2016, two enzymes capable of degrading Polyethylene terepthtalate (PET) , one of the most common plastic polymers,...

Cited by 7 publications

References 68 publications

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity

Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations

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

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