Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate

Abstract

The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus α/β-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic α/β-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.

Computational redesign of a PETase for plastic biodegradation by the GRAPE strategy

“…Although in our crystal structure W159H formed hydrogen bonding with the backbone oxygen atom of H237, molecular dynamics (MD) simulations has revealed a new hydrogen bonding formed between W159H and Ser160, whereas the original catalytic residue His237 may flip "up" out of the catalytic triad to play an aromatic stabilization role with PET ( Figure 5A). The reconstruction of the catalytic triad in the active site was also suggested by Austin et al 15 . For I168R and S188Q mutations, in addition to the new salt-bridge interaction formed between I168R and D186, the guanidine group is also suggested to donate new hydrogen bonds to the amide oxygen and backbone oxygen atoms of S188Q in the MD simulations for 10.36% of the trajectory to stabilize the enzyme structure.…”

“…Not content with the finite repertoire of naturally occurring enzymes, numerous research groups have concerned on the engineering of IsPETase, which has been summarized by Taniguchi recently 14 . Recently crystal structures of PET degrading enzymes allow for expedients that exploit rational design to improve the PET degradation activity [15][16][17][18][19][20][21] . The successful single point mutations afford 1.2 to 3.1 folds higher affinity to PET [18][19][20][21][22] , and very recently, Kim's group has succeeded in creating IsPETase S121E/D186H/R280A variant with enhanced thermal stability by 8.81 °C and 14-fold higher PET degradation activity at 40 °C 12 .…”

Bio-upcycling of polyethylene terephthalate

“…In 2016 the bacterium Ideonella sakaiensis was reported to degrade amorphous PET when cultured in the presence of yeast extract as an additional carbon source 15 . The molecular basis of the ester-bond hydrolyzing PETase and mono- (2-hydroxyethyl)TA (MHET)ase enzymes of this strain was reported in several publications (e.g., 16,17 ).…”

The modular structure of α/β‐hydrolases

“…Structures lacking the α/β‐hydrolase fold were discarded, and the remaining centroid sequences were reclustered by USEARCH with an identity threshold of 60% to reduce the number of queries for later BLAST searches. In addition, the sequences of three recently identified structures of MHETases, which also belong to the family of α/β‐hydrolases, were added to the centroids . Each centroid served as a seed sequence of a homologous family and was assigned to a superfamily , which was named by its respective architecture.…”