Mutational analysis of cutinase-like enzyme, Cut190, based on the 3D docking structure with model compounds of polyethylene terephthalate

Abstract

The cutinase-like enzyme, Cut190, from Saccharomonospora viridis AHK190 can degrade the inner block of polyethylene terephthalate (PET) in the presence of Ca²⁺, and its mutant, S226P/R228S, exhibited increased activity and higher thermostability. The crystal structures of the Cut190 S226P mutant in the absence and presence of Ca²⁺ were determined, and revealed the large conformational change induced upon Ca²⁺ binding. However, the substrate-bound 3D structures of Cut190 remained unknown. In this study, to determine the substrate-binding site and improve the enzyme activity, we first built 3D structures of a PET model compound bound to the crystal structures, using the distance restraints between the scissile carbonyl group of the compound and the catalytic site of the enzyme. We then mutated the putative substrate-binding site predicted from the models, and experimentally determined the enzymatic activities of the mutants for the model substrate poly(butylene succinate-co-adipate). The mutated sites with decreased activity were consistent with the putative binding sites predicted by the 3D model from the Ca²⁺-bound crystal structure, suggesting that the structure of the Ca²⁺-bound state represents the active state. Notably, we generated two mutants with significantly increased activities.

Keywords: Enzyme activity; Modeling; Mutation; Polyethylene terephthalate; Substrate-binding site.