Folding thermodynamics of PET-hydrolyzing enzyme Cut190 depending on Ca2+ concentration

The enzyme, cutinase from Saccharomonospora viridis AHK190 (Cut190), can hydrolyze the inner block of polyethylene terephthalate (PET). Cut190 has a unique feature that both its activity and thermal stability are increased upon Ca2+ binding. In consideration of the glass transition temperature of PET, which is between 60 and 65 degrees C, the increased activity and thermal stability are of great interest to apply for PET bio-recycling. Our previous mutational analysis showed that the S226P/R228S mutant (Cut190*) has a higher activity and thermal stability than those of the wild type. In this study, we analyzed the folding thermodynamics of the inactive mutant of Cut190*, Cut190*S176A, using circular dichroism and differential scanning calorimetry. The results show that the denaturation temperature increases from 54 to 71 degrees C due to the addition of 250mM Ca2+, in a Ca2+ concentration-dependent manner. The increased thermal stability is mainly due to the increased enthalpy change, partially compensated by the increased entropy change. Based on the crystal structure of Cut190*S176A bound to Ca2+, molecular dynamics simulations were carried out to analyze the effects of Ca2+ on the structural dynamics, showing that the Ca2+-bound structure fluctuated less than the Ca2+-free structure. Structural analysis indicates that Ca2+ binding increases the intramolecular interactions of the enzyme, while decreasing its fluctuation, which are in good correlation with the experimental results of the folding thermodynamics.