Charlier_2022_Biophys.J_121_2882

Plastic environmental pollution is a major issue that our generation must face to protect our planet. Plastic recycling not only has the potential to reduce the pollution but also to limit the need for fossil fuel-based production of new plastics. Enzymes capable of breaking down plastic could thereby support such a circular economy. Poly-ethylene terephthalate (PET) degrading enzymes have recently attracted considerable interest and were subjected to intensive enzyme engineering to improve their characteristics. A quadruple mutant of Leaf-branch Compost Cutinase (LCC) was identified as a most efficient and promising enzyme (Tournier et al., Nature 2020). Here, we use Nuclear Magnetic Resonance (NMR) to follow the initial LCC enzyme through its different mutations that lead to its improved performance. We experimentally define the two calcium binding sites and show their importance on the all-or-nothing thermal unfolding process, which occurs at a temperature of 72 degreesC close to the PET glass transition temperature. Using various NMR probes such as backbone amide, methyl group and histidine side chain resonances, we probe the interaction of the enzymes with mono-(2-hydroxyethyl)terephthalic acid (MHET). The latter experiments are interpreted in terms of accessibility of the active site to the polymer chain. STATEMENT OF SIGNIFICANCE: Plastic pollution is a persistent challenge worldwide. The PET polymer, used for plastic bottles, bags and textiles, is not easily degraded. Novel processes aimed at not only destroying the polymer but truly recycling it in a form that gives access again to the same high-quality plastics are needed. Biobased methods relying on enzymes that can depolymerize the PET might constitute an alternative to chemical catalysts to fully recover the monomers needed for renewed production of high quality PET. Here, we follow by solution NMR spectroscopy the LCC enzyme through its four mutations that turn it into a PETase that outperforms all other enzymes so far in a close-to-industrial process, and provide an experimental basis for understanding its improved characteristics.