Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET‐consuming bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended β8‐α6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in‐depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure-function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.