Polyethylene terephthalate degrading hyrdrolyase enzyme from Ideonella Sakaiensis

Meheret Ourgessa '23 and Beimnet Kassaye '23

Contents:

I. Introduction





Polyethylene terephthalate (PET) is the most abundant type of plastic in the world. It is used to make single-use bottles, packaging, containers, and clothing. PET degrades at a very slow rate under normal conditions, and this property has made it a major pollutant to both land and water. Chemical recycling is the primary way of degrading and depolymerizing PET, but the process is not cost-effective since raw PET is cheaper than its recycled counterpart.

Analysis of PET contaminated matter from a recycling plant led to the discovery of a bacterial species that uses PET as a carbon source. The bacterium, Ideonella sakaiensis, secrets the enzyme polyethylene terephthalate hydrolase (PETase) to hydrolyze the ester bond in PET and convert it to BHET (Bis(2-hydroxyethyl) terephthalate) and MHET (Mono-(2-hydroxyethyl)terephthalic acid). The bacteria also produces MHETase, an enzyme that further breaks down MHET into the monomers terephthalic acid and ethylene glycol.

While PETase from Ideonella sakaiensis has common structural and functional properties with other hydrolases (such as cutinases and lipases), it also has special features that allow it to act on PET at room temperature. Research on the mechanism of the enzymatic action is still under study, but it has already led to the discovery of mutants which degrade PET with higher efficiency.


 

Figure 1. Scheme of polyethylene terephthalate degradation by PETase into BHET(Bis(2-hydroxyethyl) terephthalate) and MHET(Mono-(2-hydroxyethyl)terephthalic acid). MHETase further breaks down MHET into the monomers TPA(terephthalic acid) and EG(ethylene glycol).

II. General Structure

A member of the α/β-hydrolase family, PETase has a core containing eight and six in its tertiary structure. The protein adopts an α/β-hydrolase fold as would be predicted from its sequence homology with proteins from the cutinase and lipase families. The surface of PETase is polarized to highly acidic and basic areas with an isoionic point of 9.6. The weight of the protein is 31.46 kDa with a residue count of 265.

Within the active-site, the catalytic triad conserved across cutinases and lipases is also present in PETase. In this protein, comprise the catalytic triad. These catalytic residues reside on loops. The nucleophilic serine residue occupies a highly conserved position called the nucleophilic/catalytic elbow. This structure has the consensus sequence (G-X1-S-X2-G). While X1 in cutinases and lipases is usually occupied with phenylalanine or histidine, in PETase, the position is occupied by tryptophan giving the enzyme an extended hydrophobic surface next to the active site.

Compared to its closest cutinases homologs, PETase has a broader active-site cleft, and, at its broadest point, approaches three times the width of the corresponding structure in the Thermobifida fusca cutinase, another enzyme known for its ability to hydrolyze PET. This broadening happened with minimal rearrangement of adjacent loops and the secondary structure; only a single amino acid substitution from phenylalanine to serine (ser160) in the active site seems to have caused it. The of PETase is then formed between two tryptophan residues (Trp159 and Trp185) and the novel Ser160.

PETase has two disulfide bonds, one that is adjacent to the active site and another one close to the C terminus of the protein. The first disulfide bond is between and connects , stabilizing the whole molecule. PETase's second disulfide bond, , is next to the active site and a hidden site in β-sheet 7, which is found immediately below the active site, to a loop that connects β-Sheet 8 with α-Helix 5. This loop has the catalytic histidine His237. The disulfide bond anchors the loop and stabilizes the histidine and the entire catalytic triad. This stabilization allows for higher flexibility at room temperature, which is a property absent in other thermophilic hydrolases.

III. PET Binding and Degradation Mechanism

IFD (Induced-fit Docking) protocol has been used to predict PET-PETase binding modes. It is thought that in a productive PET-binding event, Ser160 is positioned at a distance of 5.1 Å from the carbonyl carbon of PET. Within the same model, His237, positioned 3.9 Å from Ser160, would the serine residue for nucleophilic attack through a hydrogen bond. Further, Asp206 provides to His237 from a distance of 2.8 Å.

A of PETase bound to a ligand, HMET (1-(2-hydroxyethyl) 4-methyl terephthalate), elucidates the exact mechanism of PET degradation. HMET is a substrate analog here since, like PET, it has an ester bond moiety and an aryl group. This structure contains an to inactivate the enzyme and determine the ligand-bound structure. PET degradation seems to be achieved by the hydroxyl group of the activated Ser160 attacking the carbonyl carbon of HEMT (and, thus, likely PET too) in a nucleophilic acyl substitution mechanism. During this attack, the ester group approaches the enzyme with the carbonyl oxygen facing the formed by Tyr87 and Met161 (Figure 2). These residues are both within of the carbonyl carbon being attacked in HMET. The positively charged nitrogen atoms in the oxyanion hole can stabilize the partial negative charge on the carbonyl oxygen of the ester. Leu208 and Met161 further provide to HMET.

, a part of the active-site cleft, rotates as PET is bound in order to form aromatic interactions with the molecule. The reorientation of Trp185 in the ligand-bound model of PETase also suggests that the movement of this residue opens up the active-site of the enzyme. Additionally, seems to allow this rotation or wobbling of Trp185 and is therefore an important feature of PETase.

After the first nucleophilic attack by the activated Ser160, a water molecule will complete the cleavage of the ester bond (now between Ser160 and the substrate) which leads to the formation of tereaphtalic acid. The aromatic portion of the terephthalic acid moiety then strengthens its hydrophobic interactions (base stacking) with the indole ring in Trp185. This base stacking will induce an angular change to the conformation of the substrate initiating product release (Figure 2).

 

Figure 2. Binding and degradation of the analogous substrate HMET by PETase. Interactions with the protein during product release are also shown. All amino acid position numbers in this figure are shifted down by 29 (for example, Y58 is the same as the residue Tyr87 presented in the text).

IV. Improving Degradation

Specific mutations to the amino acids of PETase enhance degradation. A double mutant with S238F/W159H outperforms the wild type at degrading PET that has higher crystallinity like the one found in plastic bottles. Like cutinases, the mutant had a narrower active site that gave it better crystallinity reduction and product release. The S238F mutation introduced a ring that allows for more hydrophobic interactions with the surrounding terephthalate, and the W159H substitution reduced steric hindrance and deepened the space in the active site for PET binding.

Another substitution L117F was made at a position outside of the active site channel. The change from the aliphatic to the aromatic phenylalanine allowed for faster PET binding and release of products. This mutant also outperformed the wild type PETase; its degradation rate was 2.1 times that of the wild type. Mutation of , found close to the active site, to the ringed phenylalanine gave better PET binding, and the rate of degradation increased by 2.5 folds. The aromaticity of phenylalanine enhances degradation because ring stacking between the amino acid residue and terephthalate increases binding of the enzyme to PET.

The improved degradation by these protein-engineered mutants is evidence that the activity of PETase could be enhanced and more PET could be recycled faster.


V. References

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Chen, C. C., Han, X., Ko, T. P., Liu, W., & Guo, R. T. (2018). Structural studies reveal the molecular mechanism of PETase. The FEBS Journal , 285(20), 3717�3723. https://doi.org/10.1111/febs.14612

Fecker, T., Galaz-Davison, P., Engelberger, F., Narui, Y., Sotomayor, M., Parra, L. P., & Ram�rez-Sarmiento, C. A. (2018). Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase. Biophysical Journal , 114(6), 1302�1312. https://doi.org/10.1016/j.bpj.2018.02.005.

Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., � Yuan, Y. (2018). Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering. Engineering, 4(6), 888�893. https://doi.org/10.1016/j.eng.2018.09.007

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

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