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.

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Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear.H erein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. Ah ydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases,l eading to the discovery of five novel promiscuous acyltransferases.W ea lso developed as imple yet versatile colorimetric assayfor rapid characterization of novel acyltransferases.T his study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to avast number of novel acyltransferases with diverse substrate specificity and potential applications.

Sugar O-methylation shields algal polysaccharides against microbial hydrolytic enzymes. Here, we describe cytochrome P450 monooxygenases from marine bacteria that, together with appropriate redox-partner proteins, catalyze the oxidative demethylation of 6-O-methyl-D-galactose, which is an abundant monosaccharide of the algal polysaccharides agarose and porphyran. This previously unknown biological function extends the group of carbohydrate-active enzymes to include the class of cytochrome P450 monooxygenases.

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