Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation

Abstract

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 Å 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.

Structural studies reveal the molecular mechanism of PETase

“…DS2, which links the C-terminal loop and the last helix, is strictly conserved in all homologous structures [9]. This extended loop is proposed to yield a subsite to bind PET, while shorter loops found in homologous enzymes may hinder the subsite formation [12]. 3A).…”

“…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.…”

“…In spite of the many structures of PET-hydrolyzing enzymes determined by X-ray crystallography, only a few complex structures with PET or its analogs have been reported [11][12][13][14][20][21][22] and so the PET-binding pattern could only be surmised through molecular docking [11,12,14,18]. Recently, we reported the complex structures of substrate (1-(2-hydroxyethyl) 4methyl terephthalate, HEMT) and product analogs (pNP) of PET, which revealed the substrate-binding mode and allowed us to propose a mechanism of action of PETase [9].…”

“…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.…”