Substrate Report for: HEMT

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral alpha/beta-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used nowadays. Unfortunately, the polymers accumulate in nature and until now, no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly alpha/beta-hydrolases like cutinases and related enzymes (E.C. 3.1.-). Currently, only a small number of such enzymes are well characterized. Within this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 GB of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the IMG data base detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. Thereby, two novel and thermostable enzymes with high potential for downstream application were in part characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phylum of Actinobacteria, Proteobacteria and Bacteroidetes Within the Proteobacteria, the Beta-, Delta- and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum of the Bacteroidetes appear to be the main host of PET hydrolase genes rather than Actinobacteria or Proteobacteria as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although few PET hydrolases are already known it is still unknown how frequent they appear and which main bacterial phyla they are affiliated with. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed are occurring at very low frequencies in the environment. Further it was possible to link them to phyla which were previously unknown to harbor such enzymes. This work contributes novel knowledge to the phylogenetic relationship, the recent evolution and the global distribution of PET hydrolases. Finally, we describe biochemical traits of four novel PET hydrolases.

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral alpha/beta-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used nowadays. Unfortunately, the polymers accumulate in nature and until now, no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly alpha/beta-hydrolases like cutinases and related enzymes (E.C. 3.1.-). Currently, only a small number of such enzymes are well characterized. Within this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 GB of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the IMG data base detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. Thereby, two novel and thermostable enzymes with high potential for downstream application were in part characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phylum of Actinobacteria, Proteobacteria and Bacteroidetes Within the Proteobacteria, the Beta-, Delta- and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum of the Bacteroidetes appear to be the main host of PET hydrolase genes rather than Actinobacteria or Proteobacteria as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although few PET hydrolases are already known it is still unknown how frequent they appear and which main bacterial phyla they are affiliated with. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed are occurring at very low frequencies in the environment. Further it was possible to link them to phyla which were previously unknown to harbor such enzymes. This work contributes novel knowledge to the phylogenetic relationship, the recent evolution and the global distribution of PET hydrolases. Finally, we describe biochemical traits of four novel PET hydrolases.

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.

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.

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

We determined the crystal structure of a cutinase from Thermobifida alba AHK119 (Est119) at a resolution of 1.76A. The overall structure of Est119 displays a typical alpha/beta-hydrolase fold consisting of a central twisted beta-sheet of nine beta-strands that are flanked by nine alpha-helices on both sides. The refined model contains two monomers in the asymmetric unit that form a dimer interface; a polyethylene glycol fragment is bound in the interface. Polyethylene glycol-binding site on the protein may suggest a glycol-binding site. A putative polymer-recognizing groove is observed to continue through the catalytic pocket. Water molecules are bound to hydrophilic amino acids along the groove, indicating the alternating pattern of polar and hydrophobic residues.