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

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

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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:

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