Structural basis for Ca2+-dependent catalysis of a cutinase-like enzyme and its engineering: application to enzymatic PET depolymerization

Abstract

A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, can depolymerize polyethylene terephthalate (PET). As high activity at approximately 70°C is required for PET depolymerization, structure-based protein engineering of Cut190 was carried out. Crystal structure information of the Cut190 mutants was used for protein engineering and for evaluating the molecular basis of activity and thermal stability. A variety of biophysical methods were employed to unveil the mechanisms underlying the unique features of Cut190, which included the regulation of its activity and thermal stability by Ca2+. Ca2+ association and dissociation can change the enzyme conformation to regulate catalytic activity. Weak metal-ion binding would be required for the naïve conformational change of Cut190, while maintaining its fluctuation, to “switch” the enzyme on and off. The activity of Cut190 is regulated by the weak Ca2+ binding to the specific site, Site 1, while thermal stability is mainly regulated by binding to another Site 2, where a disulfide bond could be introduced to increase the stability. Recent results on the structure-activity relationship of engineered Cut190 are reviewed, including the application for PET depolymerization by enzymes.

Keywords

• crystal structure
• metal-ion binding
• polyethylene terephthalate
• thermal stability