0428同步年報-2021-全

042 ACTIVITY REPORT 2021 Fig. 2 : Structural and functional roles of Is PETase-unique Ser/Ile duo. (a) Crystal structures of Is PETase and Bur PL. Critical residues and PET substrate analogue 1-(2-hydroxyethyl) 4-methyl terephthalate (HEMT) are shown and displayed as sticks. The conformation of W185 and its equivalent in Bur PL are noted. (b) The PET- hydrolytic activity of wild-type and mutant enzymes. The MHET and TPA released in each reaction were determined and shown. Is PETase DM carries a His/Phe duo, whereas Pb PL DM and Bur PL DM carry a Ser/Ile duo. [Reproduced from Ref. 6] suitable for PET binding ( Fig. 1(c) ). The research team identified from GenBank several previously uncharacterized Is PETase-homologous cutinases that share these two features in an attempt to seek more potent PET hydrolytic enzymes. The recombinant proteins of several enzymes were obtained, but they all display PET hydrolytic activity much less than that of Is PETase. In addition to the extra disulfide bond and the loop, the research team found that the substrate-binding pocket of Is PETase is more flexible than that of other cutinases. This condition is attributed to the wobbling conformations of a TPA moiety-binding residue W185. 4 In the apo -form structure, three conformations of W185 were observed, denoted types A, B and C ( Fig. 2(a) , left panel). In the substrate analogue-bound Is PETase, W185 adopts “type-B” conformation ( Fig. 2(a) , middle panel). This effect attracted the research team's attention as W185 is strictly conserved in cutinases but they all display a “type-C” conformation ( Fig. 2(a) , right panel), which is an unfavourable PET- binding pose. On comparing Is PETase and canonical cutinase structures, the research team found that a His residue is located beneath the W185-corresponding residue in canonical cutinases, of which the imidazole side chain packs against the indole side chain and prevents Trp from swinging ( Fig. 2(a) , right panel). Significantly, the His residue is strictly conserved in all cutinases. In Is PETase, this His residue is replaced with a smaller residue Ser (S214), such that W185 is not packed and is free to swing ( Fig. 2(a) , left and middle panel). Mutagenesis experiments showed that the PET-hydrolytic activity of the Is PETase S214H variant is decreased by about half. The research team thus considered that a His-to-Ser mutation is the key to transform a canonical cutinase to a specified PET hydrolase, and introduced a His-to-Ser mutation in other Is PETase-homologous cutinases in an attempt to obtain more novel PET hydrolases. Once again, no His-to-Ser variant showed an elevated PET hydrolytic activity. The research team also solved the crystal structure of an Is PETase-homologous enzyme from Burkholderiales bacterium, denoted Bur PL. 6 The overall structure is highly identical to that of Is PETase, but the β6-β7 loop that accommodates the W185-corresponding residue of Bur PL is less flexible than that of Is PETase. In Bur PL, the side chain of a Phe residue that is located beneath the W185- corresponding residue propels the Trp Cα to maintain the β6-β7 loop in position. In Is PETase, the Phe-corresponding residue is replaced with Ile (I218), of which the side chain is smaller than the phenyl group of Phe such that the spatial hindrance of the W185-locating β6-β7 loop is relieved. The research team was amazed to find that the Ile residue is also unique to Is PETase; the corresponding residue in other homologous enzymes is Phe ( Fig. 2(a) ). The research team thus introduced His-to-Ser and Phe-to-Ile double mutation (DM) in Bur PL and found that the variant showed a three-fold increase in PET-hydrolytic activity. For another Is PETase-like cutinase from Polyangium brachysporum ( Pb PL), introducing DM enhanced its PET-hydrolytic activity by ~tenfold ( Fig. 2(b) ). The research team also introduced DM in five cutinases that are more phylogenetically distant from Is PETase, of which four showed a significantly increased PET-hydrolytic activity, including two that lacked the Is PETase unique extra disulfide bond and loop. These results indicate that Ser/ Ile DM might be a minimal requirement to transform a canonical cutinase to a specified PET hydrolase; the predecessor of Is PETase might acquire Ser/Ile DM to increase the flexibility of the substrate- binding pocket to increase the binding