Towards site-specific information on PET degrading enzymes using NMR near operational temperature

Abstract PETases are enzymes that can break down the poly-ethylene terephthalate (PET) polymer in its constituent building blocks. This enzymatic recycling process offers a sustainable solution for producing new, high-quality plastics from previously used materials. NMR spectroscopy can help in understanding and ultimately improving these enzymes but is always confronted with the lengthy step of acquisition and interpretation of triple resonance spectra for the spectral assignment. Here, we explore whether this step can be made more efficient by recording the spectra directly at high temperature, which simultaneously corresponds to more realistic working conditions for the enzyme. Taking the inactive variant of LCCICCG as an example, we compare spectral quality at 30°C and 50°C, and find that the latter condition greatly improves the Signal-to-Noise (S/N) ratio of the standard triple resonance spectra. Going up to 60ºC, we show that pulse sequences mainly used for the assignment of intrinsically disordered proteins (IDPs) also become feasible. As a result, we present a methodology enabling exhaustive backbone assignment based on a minimal set of triple resonance spectra acquired and analysed in less than two weeks. The assignment process hence can be completed on a time scale comparable to crystallography, bringing NMR in a favourable position to contribute to bio-structural studies on this family of highly thermostable PETases. Competing Interest Statement The authors have declared no competing interest. Footnotes In the new version of the manuscript we have made significant changes from the original paper to make it more general for thermostable enzymes. While the original version contained information for the assignment of complete backbone and side-chain assignments we focused on the backbone and show that in PETase this step can be speed-up.