Enhancing the kcat of IsMHETase through a single active site mutation

Enzymatic PET-recycling offers an environmentally friendly method for regenerating high quality PET from waste PET. This method relies on PET hydrolases to cleave PET into the final monomers terephtalic acid (TPA) and ethylene glycol (EG) for subsequent re-synthesis. Although PET hydrolases have been significantly improved through engineering, the final breakdown of the intermediate mono(2-hydroxyethyl) terephtalate (MHET) to TPA and EG is slow. Therefore, a dual enzyme process may be beneficial, in which PET hydrolases are accompanied by an efficient MHET hydrolysing enzyme, such as the bacterial IsMHETase from Ideonella sakaiensis. In this study we aimed to improve the catalytic efficiency of IsMHETase by altering the electrostatics in the active site. Guided by prior research we made a series of 36 variants of which ten were purified and tested for MHET hydrolysis. One variant, F495H, showed a 3.4 fold increased kcat 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 Asp492. This leads to higher polarization of the Asp492-His528 dyad and stronger electrostatic stabilization of the transition state. The discovery of this mutation provides a promising route toward faster IsMHETase variants for dual-enzyme systems with an efficient PET-hydrolase. Furthermore, this study provides insight into the structure and mechanistic features of active-site mutations that confer beneficial effects on catalysis.