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.
Widespread
utilization of polyethylene terephthalate (PET) has
caused a variety of environmental and health problems; thus, the enzymatic
degradation of PET can be a promising solution. Although PETase from Ideonalla sakaiensis (IsPETase)
has been reported to have the highest PET degradation activity under
mild conditions of all PET-degrading enzymes reported to date, its
low thermal stability limits its ability for efficient and practical
enzymatic degradation of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy
using several IsPETase variants that were screened
for high thermal stability to improve PET degradation activity. In
particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7
connecting loop and extended subsite IIc, had a T
m value that was increased by 8.81 °C and PET degradation
activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications
were further verified through structure determination of the variants,
and high thermal stability was further confirmed by a heat-inactivation
experiment. The proposed strategy and developed variants represent
an important advancement for achieving the complete biodegradation
of PET under mild conditions.
Monohydroxyethyl terephthalate (MHET) hydrolase (MHE-Tase) is an enzyme known to be involved in the final degradation step of poly(ethylene terephthalate) (PET) by hydrolyzing MHET into terephthalic acid and ethylene glycol in Ideonella sakaiensis. Here, we report the extracellular production of MHETase in an active form with a proper folding. Based on the structural observations and biochemical experiments, we reveal that MHETase also functions as exo-PETase by hydrolyzing the synthesized PET pentamer. We further present that MHETase has a hydrolysis activity against the termini-generated PET film, demonstrating the exo-PETase function of the enzyme. We also develop a MHETase R411K/S416A/F424I variant with a higher BHET activity, and the variant exhibits an enhanced degradation activity against the PET film. Based on these results, we propose that MHETase plays several roles in the biodegradation of PET using the BHETase and exo-PETase activities as well as the MHET hydrolysis function.
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