Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model
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Author(s)
Zhong-Johnson, En Ze Linda
Dong, Ziyue
Canova, Christopher T
Destro, Francesco
Cañellas, Marina
Hoffman, Mikaila C
Maréchal, Jeanne
Johnson, Timothy M
Zheng, Maya
Schlau-Cohen, Gabriela S
Date Issued
March 2024
Journal
Journal of Biological Chemistry
Publisher
Elsevier BV
Citation
Zhong-Johnson, En Ze Linda, Dong, Ziyue, Canova, Christopher T, Destro, Francesco, Cañellas, Marina et al. 2024. "Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model." Journal of Biological Chemistry, 300 (3).
Version
Final published version
Abstract
Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology. Department of Chemistry
Massachusetts Institute of Technology. Plasma Science and Fusion Center
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
https://doi.org/10.1016/j.jbc.2024.105783
