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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Tyrosinases (TYRs) catalyze two-step consecutive oxidation reactions of monophenolic compounds. Since known TYRs have optimal pH at neutral to somewhat basic pH, they have limitations to be used for production of catechol derivatives. In this study, we identified TYR from Burkholderia thailandensis (BtTYR), which exhibited high tyrosinase activity at low pH. We determined the crystal structure of BtTYR and provided the structural basis for the regulation of its activity in response to pH change. At high pH, BtTYR is inactivated due to the tight binding of its TYR and CAP domains, although it is stable in monomer form; at low pH, however, the protein is activated by the typical opening of the CAP domain, and the formation of tetramers maintains the stability of the protein. Such unique tyrosinase activity of BtTYR at acidic pH was successfully applied to highly efficient production of catechol derivatives and fabrication of an adhesive hydrogel.

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