Reply to “conformational fitting of a flexible oligomeric substrate does not explain the enzymatic pet degradation”

Reply to “conformational fitting of a flexible oligomeric substrate does not explain the enzymatic pet degradation”

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REPLYING TO Wei et al. _Nature Communications_ https://doi.org/10.1038/s41467-019-13492-9 (2019) The manuscript entitled “Conformational fitting of a flexible oligomeric substrate does not explain the enzymatic PET degradation” by Wei et al.1 raises a question on the conclusion reached in our published work, particularly regarding the docking calculations of PET substrate into the PETase from _Ideonella sakaiensis_ (_Is_PETase)2. The authors showed the ethylene glycol torsion angle _Ψ_ in an amorphous PET material of 0.25 mm thickness (Goodfellow Cambridge Ltd.) using solid-state nuclear magnetic resonance experiments and determined _trans_/_gauche_ ratio of 9:91 at 30 °C in good agreement with the previous report on amorphous PET (14 ± 5%)3. Based on the result, they suggested that the conformation of the docked 2-HE(MHET)4 in our published work that showed a _trans_ content of ethylene glycol higher than 25% is rarely present in amorphous PET polymer chains, and claimed that the residues in subsite IIb and IIc we suggested are unlikely to interact with the two MHET moieties of 2-HE(MHET)4. To support the latter statement, they further demonstrated that the transition from _gauche_ to _trans_ conformation is highly restricted in amorphous PET at 30 °C by magic-angle-spinning nuclear magnetic resonance method. They also suggested that, instead of the perfect accommodation, the key factor facilitating the substrate binding seems to be the fragile contact between the phenylene moieties and the surrounding hydrophobic residues. In general, we agree that the comments by Wei et al. provide concrete experimental results showing that ethylene glycol units of amorphous PET polymer do not have the free rotational properties to fit into its substrate binding site as the form of 2-HE(MHET)4 at 30 °C. Thus, the study provides the scientific community with useful information on PET degradation by _Is_PETase. However, our previous docking calculations2 were independent of temperature setting and not restricted to the temperature of 30 °C. Although _Is_PETase cannot maintain its activity at high temperature due to its low thermal stability, efforts to increase thermal stability of the enzyme have already been reported4. Thus, an engineered _Is_PETase with high thermal stability might be able to accommodate the PET substrate in the manner we presented at temperatures higher than 30 °C as the _trans_ content of the material was increased to 56% at 70 °C1. Moreover, we did not use the _Goodfellow_ amorphous PET in our published works2,4, which counters the comment that _Is_PETase showed almost “no” activity against crystalline PET polymer1. It was also previously shown that the semi-crystalline PET material exhibits much higher _trans_ conformation of ethylene glycol than amorphous one at ambient temperature3. Thus, we believe that the authors’ claims regarding the residues in subsite IIb and IIc may not apply under all conditions. In sum, while we agree that Wei et al. provide useful evidences that the used docking calculation is not suitable in amorphous PET at low temperature such as 30 °C, their observations are not necessarily incompatible with the general findings of our previous work2. We anticipate that Wei et al.’s work and our own will inspire future studies aimed at unraveling the exact mechanisms of PET degradation. DATA AVAILABILITY Data supporting the findings of this study are available within the article and from the corresponding author upon reasonable request. REFERENCES * Wei, R. et al. Conformational fitting of a flexible oligomeric substrate does not explain the enzymatic PET degradation. _Nat_. _Commun_. (2019). * Joo, S. et al. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. _Nat. Commun._ 9, 382 (2018). Article  ADS  Google Scholar  * Schmidt-Rohr, K., Hu, W. & Zumbulyadis, N. Elucidation of the chain conformation in a glassy polyester, PET, by two-dimensional NMR. _Science_ 280, 714–717 (1998). Article  ADS  CAS  Google Scholar  * Son, H. F. et al. Rational protein engineering of thermo-stable PETase from _Ideonella sakaiensis_ for highly efficient PET degradation. _ACS Catal._ 9, 3519–3526 (2019). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT (MSIT) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF2012M1A2A2026557). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Life Sciences (KNU Creative BioResearch Group), KNU Institute for Microorganisms, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu, 41566, Republic of Korea Hogyun Seo, Seongjoon Joo, Hyeoncheol Francis Son, Hye-Young Sagong & Kyung-Jin Kim * Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare Cross Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea In Jin Cho, So Young Choi & Sang Yup Lee * BioProcess Engineering Research Center and BioInformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea Sang Yup Lee Authors * Hogyun Seo View author publications You can also search for this author inPubMed Google Scholar * In Jin Cho View author publications You can also search for this author inPubMed Google Scholar * Seongjoon Joo View author publications You can also search for this author inPubMed Google Scholar * Hyeoncheol Francis Son View author publications You can also search for this author inPubMed Google Scholar * Hye-Young Sagong View author publications You can also search for this author inPubMed Google Scholar * So Young Choi View author publications You can also search for this author inPubMed Google Scholar * Sang Yup Lee View author publications You can also search for this author inPubMed Google Scholar * Kyung-Jin Kim View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.S., S.Y.C., S.Y.L., and K.-J.K. wrote the initial paper. H.S., I.J.C., S.J., H.F.S., H.-Y.S., S.Y.C., S.Y.L., and K.-J.K. finalized the paper. CORRESPONDING AUTHORS Correspondence to Sang Yup Lee or Kyung-Jin Kim. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Seo, H., Cho, I.J., Joo, S. _et al._ Reply to “Conformational fitting of a flexible oligomeric substrate does not explain the enzymatic PET degradation”. _Nat Commun_ 10, 5582 (2019). https://doi.org/10.1038/s41467-019-13493-8 Download citation * Received: 09 October 2019 * Accepted: 04 November 2019 * Published: 06 December 2019 * DOI: https://doi.org/10.1038/s41467-019-13493-8 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. 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REPLYING TO Wei et al. _Nature Communications_ https://doi.org/10.1038/s41467-019-13492-9 (2019) The manuscript entitled “Conformational fitting of a flexible oligomeric substrate does not


explain the enzymatic PET degradation” by Wei et al.1 raises a question on the conclusion reached in our published work, particularly regarding the docking calculations of PET substrate into


the PETase from _Ideonella sakaiensis_ (_Is_PETase)2. The authors showed the ethylene glycol torsion angle _Ψ_ in an amorphous PET material of 0.25 mm thickness (Goodfellow Cambridge Ltd.)


using solid-state nuclear magnetic resonance experiments and determined _trans_/_gauche_ ratio of 9:91 at 30 °C in good agreement with the previous report on amorphous PET (14 ± 5%)3. Based


on the result, they suggested that the conformation of the docked 2-HE(MHET)4 in our published work that showed a _trans_ content of ethylene glycol higher than 25% is rarely present in


amorphous PET polymer chains, and claimed that the residues in subsite IIb and IIc we suggested are unlikely to interact with the two MHET moieties of 2-HE(MHET)4. To support the latter


statement, they further demonstrated that the transition from _gauche_ to _trans_ conformation is highly restricted in amorphous PET at 30 °C by magic-angle-spinning nuclear magnetic


resonance method. They also suggested that, instead of the perfect accommodation, the key factor facilitating the substrate binding seems to be the fragile contact between the phenylene


moieties and the surrounding hydrophobic residues. In general, we agree that the comments by Wei et al. provide concrete experimental results showing that ethylene glycol units of amorphous


PET polymer do not have the free rotational properties to fit into its substrate binding site as the form of 2-HE(MHET)4 at 30 °C. Thus, the study provides the scientific community with


useful information on PET degradation by _Is_PETase. However, our previous docking calculations2 were independent of temperature setting and not restricted to the temperature of 30 °C.


Although _Is_PETase cannot maintain its activity at high temperature due to its low thermal stability, efforts to increase thermal stability of the enzyme have already been reported4. Thus,


an engineered _Is_PETase with high thermal stability might be able to accommodate the PET substrate in the manner we presented at temperatures higher than 30 °C as the _trans_ content of the


material was increased to 56% at 70 °C1. Moreover, we did not use the _Goodfellow_ amorphous PET in our published works2,4, which counters the comment that _Is_PETase showed almost “no”


activity against crystalline PET polymer1. It was also previously shown that the semi-crystalline PET material exhibits much higher _trans_ conformation of ethylene glycol than amorphous one


at ambient temperature3. Thus, we believe that the authors’ claims regarding the residues in subsite IIb and IIc may not apply under all conditions. In sum, while we agree that Wei et al.


provide useful evidences that the used docking calculation is not suitable in amorphous PET at low temperature such as 30 °C, their observations are not necessarily incompatible with the


general findings of our previous work2. We anticipate that Wei et al.’s work and our own will inspire future studies aimed at unraveling the exact mechanisms of PET degradation. DATA


AVAILABILITY Data supporting the findings of this study are available within the article and from the corresponding author upon reasonable request. REFERENCES * Wei, R. et al. Conformational


fitting of a flexible oligomeric substrate does not explain the enzymatic PET degradation. _Nat_. _Commun_. (2019). * Joo, S. et al. Structural insight into molecular mechanism of


poly(ethylene terephthalate) degradation. _Nat. Commun._ 9, 382 (2018). Article  ADS  Google Scholar  * Schmidt-Rohr, K., Hu, W. & Zumbulyadis, N. Elucidation of the chain conformation


in a glassy polyester, PET, by two-dimensional NMR. _Science_ 280, 714–717 (1998). Article  ADS  CAS  Google Scholar  * Son, H. F. et al. Rational protein engineering of thermo-stable PETase


from _Ideonella sakaiensis_ for highly efficient PET degradation. _ACS Catal._ 9, 3519–3526 (2019). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was


supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science and ICT (MSIT) through the National


Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF2012M1A2A2026557). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Life Sciences (KNU Creative BioResearch Group), KNU


Institute for Microorganisms, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu, 41566, Republic of Korea Hogyun Seo, Seongjoon Joo, Hyeoncheol Francis Son, Hye-Young Sagong & 


Kyung-Jin Kim * Metabolic and Biomolecular Engineering National Research Laboratory, Systems Metabolic Engineering and Systems Healthcare Cross Generation Collaborative Laboratory,


Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu,


Daejeon, 34141, Republic of Korea In Jin Cho, So Young Choi & Sang Yup Lee * BioProcess Engineering Research Center and BioInformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu,


Daejeon, 34141, Republic of Korea Sang Yup Lee Authors * Hogyun Seo View author publications You can also search for this author inPubMed Google Scholar * In Jin Cho View author publications


You can also search for this author inPubMed Google Scholar * Seongjoon Joo View author publications You can also search for this author inPubMed Google Scholar * Hyeoncheol Francis Son


View author publications You can also search for this author inPubMed Google Scholar * Hye-Young Sagong View author publications You can also search for this author inPubMed Google Scholar *


So Young Choi View author publications You can also search for this author inPubMed Google Scholar * Sang Yup Lee View author publications You can also search for this author inPubMed 


Google Scholar * Kyung-Jin Kim View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.S., S.Y.C., S.Y.L., and K.-J.K. wrote the initial paper.


H.S., I.J.C., S.J., H.F.S., H.-Y.S., S.Y.C., S.Y.L., and K.-J.K. finalized the paper. CORRESPONDING AUTHORS Correspondence to Sang Yup Lee or Kyung-Jin Kim. ETHICS DECLARATIONS COMPETING


INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and


institutional affiliations. RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,


adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons


license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a


credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted


use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT


THIS ARTICLE CITE THIS ARTICLE Seo, H., Cho, I.J., Joo, S. _et al._ Reply to “Conformational fitting of a flexible oligomeric substrate does not explain the enzymatic PET degradation”. _Nat


Commun_ 10, 5582 (2019). https://doi.org/10.1038/s41467-019-13493-8 Download citation * Received: 09 October 2019 * Accepted: 04 November 2019 * Published: 06 December 2019 * DOI:


https://doi.org/10.1038/s41467-019-13493-8 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not


currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative