Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.

The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca2+ -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca2+ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca2+ to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS.

Multiple structural states of Ca2+-regulated PET hydrolase, Cut190, and its correlation with activity and stability.

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.

Metal binding to cutinase-like enzyme from Saccharomonospora viridis AHK190 and its effects on enzyme activity and stability.

A cutinase from Saccharomonospora viridis AHK190, Cut190, can hydrolyze polyethylene terephthalate and has a unique feature that the activity and stability are regulated by Ca2+ binding. Our recent structural and functional analyses showed three Ca2+ binding sites and their respective roles. Here, we analysed the binding thermodynamics of Mn2+, Zn2+ and Mg2+ to Cut190 and their effects on the catalytic activity and thermal stability. The binding affinities of Mn2+ and Zn2+ were higher than that of Mg2+ and are all entropy driven with a binding stoichiometry of three, one and one for Zn2+, Mn2+ and Mg2+, respectively. The catalytic activity was measured in the presence of the respective metals, where the activity of 0.25 mM Mn2+ was comparable to that of 2.5 mM Ca2+. Our 3D Reference Interaction Site Model calculations suggested that all the ions exhibited a high occupancy rate for Site 2. Thus, Mn2+ and Mg2+ would most likely bind to Site 2 (contributes to stability) with high affinity, while to Sites 1 and 3 (contributes to activity) with low affinity. We elucidate the metal-dependent structural and functional properties of Cut190 and show the subtle balance on structure stability and flexibility is controlled by specific metal ions.