Substrate Report for: 2HE(MHET)4

Accelerated hydrolysis of polyethylene terephthalate (PET) by enzymatic surface modification of various hydrolases, which would not degrade the building blocks of PET in order to retain the quality of recycled PET, is a promising research area. Many studies have been reported to identify mutations of different hydrolases that can improve PET degradation. Recently, the mutation of glycine and phenyl alanine with alanine in cutinase was found to improve the activity of PET degradation 6-fold. Yet, a deep insight into the overall structural basis as well as the explicit role played by the amino acid residues for PET degradation is still elusive, which is nevertheless important for comparative analyses, structure-function relations and rational optimization of the degradation process. Our molecular dynamics simulations coupled with quantum mechanical study demonstrate that mutations of anchor residue phenyl alanine to alanine at the PET binding cleft of cutinase unveiled a distal yet novel binding subsite, which alters the nature of dispersive interaction for PET recognition and binding. The phenyl alanine engages in Pi-Pi interaction with the phenyl ring of PET (-8.5 kcal mol(-1)), which on one side helps in PET recognition, but on the other side restricts PET to attain fully extended conformations over the entire binding cleft. The loss of Pi-Pi interaction due to mutation of phenyl alanine to alanine is not only compensated by the favourable cation-Pi and hydrophobic interactions from the arginine residues (-17.1 kcal mol(-1)) found in the newly discovered subsite, but also favours the fully extended PET conformation. This subsequently impacts the overall increased catalytic activity of mutated cutinase.

Accelerated hydrolysis of polyethylene terephthalate (PET) by enzymatic surface modification of various hydrolases, which would not degrade the building blocks of PET in order to retain the quality of recycled PET, is a promising research area. Many studies have been reported to identify mutations of different hydrolases that can improve PET degradation. Recently, the mutation of glycine and phenyl alanine with alanine in cutinase was found to improve the activity of PET degradation 6-fold. Yet, a deep insight into the overall structural basis as well as the explicit role played by the amino acid residues for PET degradation is still elusive, which is nevertheless important for comparative analyses, structure-function relations and rational optimization of the degradation process. Our molecular dynamics simulations coupled with quantum mechanical study demonstrate that mutations of anchor residue phenyl alanine to alanine at the PET binding cleft of cutinase unveiled a distal yet novel binding subsite, which alters the nature of dispersive interaction for PET recognition and binding. The phenyl alanine engages in Pi-Pi interaction with the phenyl ring of PET (-8.5 kcal mol(-1)), which on one side helps in PET recognition, but on the other side restricts PET to attain fully extended conformations over the entire binding cleft. The loss of Pi-Pi interaction due to mutation of phenyl alanine to alanine is not only compensated by the favourable cation-Pi and hydrophobic interactions from the arginine residues (-17.1 kcal mol(-1)) found in the newly discovered subsite, but also favours the fully extended PET conformation. This subsequently impacts the overall increased catalytic activity of mutated cutinase.

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 A resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.