8cru

PETase Ancestral Sequence Reconstruction 008PETase Ancestral Sequence Reconstruction 008

Structural highlights

8cru is a 1 chain structure with sequence from Ideonella sakaiensis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.3Å
Ligands:
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

PETH_PISS1 Involved in the degradation and assimilation of the plastic poly(ethylene terephthalate) (PET), which allows I.sakaiensis to use PET as its major energy and carbon source for growth. Likely acts synergistically with MHETase to depolymerize PET (PubMed:26965627). Catalyzes the hydrolysis of PET to produce mono(2-hydroxyethyl) terephthalate (MHET) as the major product (PubMed:26965627, PubMed:29235460, PubMed:29374183, PubMed:29603535, PubMed:29666242, PubMed:32269349). Also depolymerizes another semiaromatic polyester, poly(ethylene-2,5-furandicarboxylate) (PEF), which is an emerging, bioderived PET replacement with improved gas barrier properties (PubMed:29666242). In contrast, PETase does not degrade aliphatic polyesters such as polylactic acid (PLA) and polybutylene succinate (PBS) (PubMed:29666242). Is also able to hydrolyze bis(hydroxyethyl) terephthalate (BHET) to yield MHET with no further decomposition, but terephthalate (TPA) can also be observed (PubMed:26965627, PubMed:29374183, PubMed:29603535). Shows esterase activity towards p-nitrophenol-linked aliphatic esters (pNP-aliphatic esters) in vitro (PubMed:26965627, PubMed:30502092).^[1] ^[2] ^[3] ^[4] ^[5] ^[6] ^[7]

Publication Abstract from PubMed

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 degrees C higher than that of IsPETase and two variants with significantly higher catalytic activity.

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity.,Joho Y, Vongsouthi V, Spence MA, Ton J, Gomez C, Tan LL, Kaczmarski JA, Caputo AT, Royan S, Jackson CJ, Ardevol A Biochemistry. 2023 Jan 17;62(2):437-450. doi: 10.1021/acs.biochem.2c00323. Epub , 2022 Aug 11. PMID:35951410^[8]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016 Mar 11;351(6278):1196-9. doi: 10.1126/science.aad6359. PMID:26965627 doi:http://dx.doi.org/10.1126/science.aad6359
↑ Han X, Liu W, Huang JW, Ma J, Zheng Y, Ko TP, Xu L, Cheng YS, Chen CC, Guo RT. Structural insight into catalytic mechanism of PET hydrolase. Nat Commun. 2017 Dec 13;8(1):2106. doi: 10.1038/s41467-017-02255-z. PMID:29235460 doi:http://dx.doi.org/10.1038/s41467-017-02255-z
↑ Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, Choi SY, Lee SY, Kim KJ. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat Commun. 2018 Jan 26;9(1):382. doi: 10.1038/s41467-018-02881-1. PMID:29374183 doi:http://dx.doi.org/10.1038/s41467-018-02881-1
↑ Liu B, He L, Wang L, Li T, Li C, Liu H, Luo Y, Bao R. Protein Crystallography and Site-Direct Mutagenesis Analysis of the Poly(ethylene terephthalate) Hydrolase PETase from Ideonella sakaiensis. Chembiochem. 2018 Mar 30. doi: 10.1002/cbic.201800097. PMID:29603535 doi:http://dx.doi.org/10.1002/cbic.201800097
↑ Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, Woodcock HL, McGeehan JE, Beckham GT. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci U S A. 2018 Apr 17. pii: 1718804115. doi:, 10.1073/pnas.1718804115. PMID:29666242 doi:http://dx.doi.org/10.1073/pnas.1718804115
↑ Liu C, Shi C, Zhu S, Wei R, Yin CC. Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis. Biochem Biophys Res Commun. 2019 Jan 1;508(1):289-294. doi:, 10.1016/j.bbrc.2018.11.148. Epub 2018 Nov 27. PMID:30502092 doi:http://dx.doi.org/10.1016/j.bbrc.2018.11.148
↑ Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux ML, Texier H, Gavalda S, Cot M, Guemard E, Dalibey M, Nomme J, Cioci G, Barbe S, Chateau M, Andre I, Duquesne S, Marty A. An engineered PET depolymerase to break down and recycle plastic bottles. Nature. 2020 Apr;580(7802):216-219. doi: 10.1038/s41586-020-2149-4. Epub 2020 Apr, 8. PMID:32269349 doi:http://dx.doi.org/10.1038/s41586-020-2149-4
↑ Joho Y, Vongsouthi V, Spence MA, Ton J, Gomez C, Tan LL, Kaczmarski JA, Caputo AT, Royan S, Jackson CJ, Ardevol A. Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity. Biochemistry. 2023 Jan 17;62(2):437-450. PMID:35951410 doi:10.1021/acs.biochem.2c00323

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OCA

[1] Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016 Mar 11;351(6278):1196-9. doi: 10.1126/science.aad6359. PMID:26965627 doi:http://dx.doi.org/10.1126/science.aad6359

[2] Han X, Liu W, Huang JW, Ma J, Zheng Y, Ko TP, Xu L, Cheng YS, Chen CC, Guo RT. Structural insight into catalytic mechanism of PET hydrolase. Nat Commun. 2017 Dec 13;8(1):2106. doi: 10.1038/s41467-017-02255-z. PMID:29235460 doi:http://dx.doi.org/10.1038/s41467-017-02255-z

[3] Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, Choi SY, Lee SY, Kim KJ. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat Commun. 2018 Jan 26;9(1):382. doi: 10.1038/s41467-018-02881-1. PMID:29374183 doi:http://dx.doi.org/10.1038/s41467-018-02881-1

[4] Liu B, He L, Wang L, Li T, Li C, Liu H, Luo Y, Bao R. Protein Crystallography and Site-Direct Mutagenesis Analysis of the Poly(ethylene terephthalate) Hydrolase PETase from Ideonella sakaiensis. Chembiochem. 2018 Mar 30. doi: 10.1002/cbic.201800097. PMID:29603535 doi:http://dx.doi.org/10.1002/cbic.201800097

[5] Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, Woodcock HL, McGeehan JE, Beckham GT. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci U S A. 2018 Apr 17. pii: 1718804115. doi:, 10.1073/pnas.1718804115. PMID:29666242 doi:http://dx.doi.org/10.1073/pnas.1718804115

[6] Liu C, Shi C, Zhu S, Wei R, Yin CC. Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis. Biochem Biophys Res Commun. 2019 Jan 1;508(1):289-294. doi:, 10.1016/j.bbrc.2018.11.148. Epub 2018 Nov 27. PMID:30502092 doi:http://dx.doi.org/10.1016/j.bbrc.2018.11.148

[7] Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux ML, Texier H, Gavalda S, Cot M, Guemard E, Dalibey M, Nomme J, Cioci G, Barbe S, Chateau M, Andre I, Duquesne S, Marty A. An engineered PET depolymerase to break down and recycle plastic bottles. Nature. 2020 Apr;580(7802):216-219. doi: 10.1038/s41586-020-2149-4. Epub 2020 Apr, 8. PMID:32269349 doi:http://dx.doi.org/10.1038/s41586-020-2149-4

[8] Joho Y, Vongsouthi V, Spence MA, Ton J, Gomez C, Tan LL, Kaczmarski JA, Caputo AT, Royan S, Jackson CJ, Ardevol A. Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity. Biochemistry. 2023 Jan 17;62(2):437-450. PMID:35951410 doi:10.1021/acs.biochem.2c00323

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