Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase

Abstract

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 Å 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.

Structural studies reveal the molecular mechanism of PETase

“…The PETase-specific DS1, on the other hand, is located adjacent to the active site and connects the b7-a5 and b8-a6 loops that harbor the catalytic acid (D177) and catalytic base (H208), respectively (Fig. Interestingly, PET hydrolysis measured under reducing conditions and mutagenesis experiments indicate that DS1 depletion significantly reduces enzyme activity, suggesting that DS1 plays a critical role in PETase activity [9,[11][12][13]. In addition, the b8-a6 loop in PETase is three residues longer than that of other homologous enzymes (Fig.…”

“…3A). Molecular dynamic simulation suggests that DS1 depletion may influence the distances between the catalytic triad residues and thus compromise the active site integrity [11]. 3A).…”

“…Significantly, I. sakaiensis secretes a unique cutinase-like enzyme, later dubbed PETase, to hydrolyze PET to bis(2-hydroxylethyl) TPA (BHET), mono(2-hydroxyethyl) TPA (MHET), and TPA ( Fig. Here, we summarize the most recent studies of crystal structures of PETase and its complex with substrate/product analogs reported by ourselves and other groups [9][10][11][12][13]. Under physiological conditions (30°C, pH 7.0), the enzyme is 5.5-to 120-fold more efficient than previously reported PET-hydrolyzing homologs.…”

“…Recently, we and other groups independently reported the crystal structure of PETase [9][10][11][12][13]. The enzyme adopts a typical a/b-hydrolase fold, containing a twisted central b-sheet consisting of nine b-strands which is sandwiched by six a-helices ( Fig.…”

Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants

“…[4][5][6][7][8][9][10][11] For example, cutinases from Thermobifida fusca [8,9] and the most efficient PET hydrolyzing esterase, leaf and branch compost cutinase( LCC). [12][13][14][15][16][17][18][19][20] Although PETase is highly specific to PET,t he enzymes hows poor catalytic activity and is difficult to use in industrial processes. [12][13][14][15][16][17][18][19][20] Although PETase is highly specific to PET,t he enzymes hows poor catalytic activity and is difficult to use in industrial processes.…”