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

• Published in: Journal of thermal analysis and calorimetry Vol. 135; no. 5; pp. 2655 - 2663
• Main Authors: Inaba, Satomi, Kamiya, Narutoshi, Bekker, Gert-Jan, Kawai, Fusako, Oda, Masayuki
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.03.2019; Springer Nature B.V
• Subjects: Analytical Chemistry; Binding; Calcium ions; Chemistry; Chemistry and Materials Science; Crystal structure; Denaturation; Dichroism; Dynamic structural analysis; Enthalpy; Enzymes; Folding; Glass transition temperature; Inorganic Chemistry; Measurement Science and Instrumentation; Molecular dynamics; Physical Chemistry; Polyethylene terephthalate; Polymer Sciences; Stability analysis; Structural analysis; Temperature; Thermal stability; Thermodynamics; Variations; Molecular dynamics simulations; DSC; Protein conformational change; Circular dichroism
• DOI: 10.1007/s10973-018-7447-9
• ISSN: 1388-6150

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 Ca 2+ binding. In consideration of the glass transition temperature of PET, which is between 60 and 65 °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 °C due to the addition of 250 mM Ca 2+ , in a Ca 2+ 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 Ca 2+ , molecular dynamics simulations were carried out to analyze the effects of Ca 2+ on the structural dynamics, showing that the Ca 2+ -bound structure fluctuated less than the Ca 2+ -free structure. Structural analysis indicates that Ca 2+ 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.