Enhancing the catalytic performance of IsMHETase through electrostatic engineering of the active site

This is the authors' abstract. We don't add key points for this paper.

Enzymatic polyethylene terephthalate (PET) recycling offers an environmentally friendly method for regenerating virgin PET from waste PET. However, a remaining obstacle is the inefficient breakdown of the smallest intermediate, mono(2-hydroxyethyl) terephthalic acid (MHET). A previously studied enzyme, Is MHETase, is of particular interest for its activity on MHET. This study aims to rationally improve the catalytic efficiency of the enzyme Is MHETase by altering the electrostatics in the active site in a series of variants. One variant, F495H, showed a 3.4-fold increased k cat and was further characterized both experimentally and computationally. pH and salt profiles showed that the enhanced turnover by F495H depends strongly on the protonation state of the introduced histidine. Computational studies indicate that F495H leaves the binding pose largely unaltered but instead modulates the local electrostatic environment of the catalytic residue Asp 492 . This study provides insight into the structure and mechanistic features of active-site mutations that confer beneficial effects on catalysis.