Structural insights into the unique polylactate‐degrading mechanism of Thermobifida alba cutinase

Abstract

Cutinases are enzymes known to degrade polyester‐type plastics. Est119, a plastic‐degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA‐degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after‐reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic‐degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering.

Enzymatic Remediation of Polyethylene Terephthalate (PET)–Based Polymers for Effective Management of Plastic Wastes: An Overview

“…Hydrolysis of synthetic polymers such as PET (Dimarogona et al, 2015), polycaprolactone (Adıgüzel and Tunçer, 2017), polystyrene (PS) (Ho et al, 2018), polyethylene furanoate (Weinberger et al, 2017), and polybutylene succinate (Hu et al, 2016) have also been reported using cutinase. Cutinase-mediated hydrolysis of polylactic acid is also demonstrated by several authors (Masaki et al, 2005;Kitadokoro et al, 2019).…”

An extracellular lipase from Amycolatopsis mediterannei is a cutinase with plastic degrading activity

“…The mechanism of PLA degradation by a cutinase from T. alba has been recently reported [40] . The cutinase ( T.alba cutinase Est119) has a sequence similar to AML (61% sequence identity, 73% sequence similarity).…”

“…It was clear from this alignment that there was significant overlap in the key catalytic residues for these enzymes. The structural differences were further explored by superimposing the 3D model of AML on the crystal structure of Est119 crystallised with bound PLA analogues ethyl acetate (EL) and lactic acid (LAC; PDB: 6AID) [40] . Both structures were aligned using PyMOL to compare the reported catalytic and substrate recognition sites for PLA degradation (see Fig.…”

“…It is unclear why these differences should account for such divergence in PLA degrading activity given the largely similar nature of the residues. Kitadokoro and co-workers postulated that Tyr99 of Est119 is important as it provides the oxyanion hole to stabilize the esterase reaction [40] . Nonetheless, from this study we can conclude that relatively minor differences in the wider substrate binding site can give rise to significant differences in substrate specificity for plastics.…”

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

“…Residue X acid+2 , one of the conserved structural elements that is involved in the coordination of the imidazole ring of the catalytic histidine, is usually a hydrophobic or an aromatic residue located at the entrance of the active site cleft, mostly reviewed for its role in ligand binding [51][52][53][54][55][56] or the release of products of catalysis [51,57]. Consequently, the site-directed mutagenesis of residue X acid+2 in different ABH enzymes has led to various results, which are consistent with its role in ligand binding, including the compromising [52,54,55,58,59] or the enhancement [52] of catalytic activity, the alteration of transport tunnels [57,60], the modification of substrate specificity [53,55,56] and the inversion of enantioselectivity [52]. For the remaining residues that coordinate the imidazole ring of the catalytic histidine, including residue X acid+3 , we have only found a few mutational studies that have resulted in reduced activity [58,61,62], with a single study highlighting the residue's role in the stability of the enzyme and the catalytic activity rather than in ligand binding [58].…”