NMR at operational temperature for resonance assignments of a PET degrading enzyme

PETases are enzymes that can break down the PET polymer in its constituent building blocks, and thereby recycle starting material for new high-quality plastics. NMR spectroscopy can help in the understanding and ultimately improvement of these PETases, 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 also corresponds to more realistic working conditions for the enzyme. Taking the inactive variant of LCCICCG in which the Serine 165 has been replaced by an Alanine (LCCICCG-S165A) as an example, we evaluate spectral quality at 30C and 50C, and find that the latter condition greatly improves the Signal-to-Noise (S/N) ratio of the different spectra. As a result, we present an exhaustive backbone and side-chain assignment of LCCICCG-S165A based on a minimal set of triple resonance spectra acquired at 50C, that can act as a basis for future work on bio-structural studies on this PETase.