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Introduction

Structural Overview

Catalytic Triad

As the PET substrate binds to the enzyme, its carbonyl bond attached to the first benzene ring must be harbored close to Serine in the catalytic triad (Fig. 2). Serine gets polarized by histidine, which is then stabilized by aspartic acid. Polarized Serine will attack the carbonyl bond (C-O) of the polyester, resulting in a tetrahedral in- termediate, which is then stabilized by the oxyanion hole. The hy- drolysis procedure is completed by a second nucleophilic attack mediated through a water molecule "PEThydrolase-insilico-engineering"

Ligand Binding Site

hydrophobic pocket from "An engineered PET depolymerase to break down and recycle plastic bottles"

Figure 1

Mutations

F243

F243W

T96

Y127

N246

S283 & D238

It gains stability with two disulfide bonds, while only one conserved disulfide bond exists in other homologs. Conserved disulfide bond C273-C289 connects the last loop to the C-terminal helix. The IsPETase-specific disulfide bond C203-C239 harbors the catalytic acid and the base [43], and has been shown to result in a lower energy barrier/higher efficiency for PET hydrolysis - "PEThydrolase-insilico-engineering"

Key residues that were on the

metal site of known homolog enzymes were identified as D238 and S283. They mutated by cysteine to introduce a disulfide bonding to increase melting temperature instead of metal ion stabilization. The engineered LCC version named LCC-ICCG (with D238C/S283C along with F243I and Y127G) is reported to acieve 90% of PET depoly- merization. Recent study by Zeng and coworkers reported an in- crease in the melting temperature of LCC-ICCG to 98.9 °C by addition of A59K, V63I, and N248P mutations. However, the optimal hydro- lyzing temperature was found to be 74 °C [58]. -"PEThydrolase-insilico-engineering"