Conformational Selection in Biocatalytic Plastic Degradation by PETase

Abstract

Due to the steric effects imposed by bulky polymers, the formation of catalytically competent enzyme and substrate conformations is critical in the biodegradation of plastics. In poly­(ethylene terephthalate) (PET), the backbone adopts different conformations, gauche and trans, coexisting to different extents in amorphous and crystalline regions. However, which conformation is susceptible to biodegradation and the extent of enzyme and substrate conformational changes required for expedient catalysis remain poorly understood. To overcome this obstacle, we utilized molecular dynamics simulations, docking, and enzyme engineering in concert with high-resolution microscopy imaging and solid-state nuclear magnetic resonance (NMR) to demonstrate the importance of conformational selection in biocatalytic plastic hydrolysis. Our results demonstrate how single-amino acid substitutions in Ideonella sakaiensis PETase can alter its conformational landscape, significantly affecting the relative abundance of productive ground-state structures ready to bind discrete substrate conformers. We experimentally show how an enzyme binds to plastic and provide a model for key residues involved in the recognition of gauche and trans conformations supported by in silico simulations. We demonstrate how enzyme engineering can be used to create a trans-selective variant, resulting in higher activity when combined with an all-trans PET-derived oligomeric substrate, stemming from both increased accessibility and conformational preference. Our work cements the importance of matching enzyme and substrate conformations in plastic hydrolysis, and we show that also the noncanonical trans conformation in PET is conducive for degradation. Understanding the contribution of enzyme and substrate conformations to biocatalytic plastic degradation could facilitate the generation of designer enzymes with increased performance.

Particle Size Reduction of Poly(ethylene terephthalate) Increases the Rate of Enzymatic Depolymerization But Does Not Increase the Overall Conversion Extent

“…Enzymatic recycling is an emerging chemical recycling strategy for PET due to the relatively low temperature and pressure required, as well as high selectivity in the enzymatic reactions, possibly enabling precise PET depolymerization within complex mixed waste streams that have been previously excluded from recycling processes. − Many PET-degrading enzymes have been previously reported and characterized, − but most show limited activity on PET and are therefore likely not directly suitable for industrial use. There have recently been many efforts to enhance the thermostability and catalytic activity of several PET hydrolases to this end. − …”

Plastic Waste Valorization by Leveraging Multidisciplinary Catalytic Technologies

“…158 To tackle this issue, enzyme engineering has significantly improved the activity and stability of PET hydrolase for biological recycling of PET to high-quality monomers (Figure 7). 20,21,160,161 Tournier and co-workers reported the computer-aided enzyme engineering of PET hydrolase, achieving 90% conversion of amorphous PET in less than 10 h with a mean productivity of 16.7 g TPA L −1 h −1 . 20 The formed monomers can be repolymerized to obtain PET that exhibits the same properties as petrochemical-produced PET.…”

Cation–π and hydrophobic interaction controlled PET recognition in double mutated cutinase – identification of a novel binding subsite for better catalytic activity

“…Earlier binding studies did not indicate PET binding to such residues. 13,14,36,[74][75][76][77] At this point we might reason that, the mutation of the aromatic Phe209 (F209) in WT to hydrophobic Ala209 (A209) in DM cutinase might have affected the conformation of PET binding on the surface of cutinase. However, further analysis is required to elucidate this striking difference in the mode of PET binding upon mutation.…”