Structure Report for: 7CTR

Emori, M.,Numoto, N.,Senga, A.,Bekker, G.J.,Kamiya, N.,Ito, N.,Kawai, F.,Oda, M.

Title: Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability
Emori M, Numoto N, Senga A, Bekker GJ, Kamiya N, Kobayashi Y, Ito N, Kawai F, Oda M
Ref: Proteins, 89:502, 2021 : PubMed
Abstract


ESTHER: Emori_2021_Proteins_89_502
PubMedSearch: Emori 2021 Proteins 89 502
PubMedID: 33340163
Gene_locus related to this paper: sacvd-c7mve8

Abstract

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 Ca(2+) -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 Ca(2+) to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca(2+) 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 degreesC, 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.