Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

CK2α and CK2α′ are paralogous catalytic subunits of CK2, which belongs to the eukaryotic protein kinases. CK2 promotes tumorigenesis and the spread of pathogenic viruses like SARS-CoV-2 and is thus an attractive drug target. Efforts to develop selective CK2 inhibitors binding offside the ATP site had disclosed the αD pocket in CK2α; its occupation requires large conformational adaptations of the helix αD. As shown here, the αD pocket is accessible also in CK2α′, where the necessary structural plasticity can be triggered with suitable ligands even in the crystalline state. A CK2α′ structure with an ATP site and an αD pocket ligand guided the design of the bivalent CK2 inhibitor KN2. It binds to CK2 with low nanomolar affinity, is cell-permeable, and suppresses the intracellular phosphorylation of typical CK2 substrates. Kinase profiling revealed a high selectivity of KN2 for CK2 and emphasizes the selectivity-promoting potential of the αD pocket.

Selective inhibitors of protein kinase CK2 with significant cytotoxicity on tumor cells based on a 2-aminothiazole scaffold were described recently. Here, these studies are supplemented with representative CK2α/CK2α′ complex structures. They reveal that the 2-aminothiazole-based inhibitors occupy the ATP cavity, whereas preliminary data had indicated an allosteric binding site. The crystal structure findings are corroborated by subsequent enzyme kinetic studies; their atomic-resolution quality provides the basis for future optimization of these promising CK2 inhibitors.

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 40 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme has been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46, an archaeal promiscuous feruloyl esterase exhibiting degradation activitiy on PET, bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, was derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Angstrom. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study significantly widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a lignin-degrading esterase.

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