Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism

Several bacteria possess components of catabolic pathways for the synthetic polyester poly(ethylene terephthalate) (PET). These proceed by hydrolyzing the ester linkages of the polymer to its monomers, ethylene glycol and terephthalate (TPA), which are further converted into common metabolites. These pathways are crucial for genetically engineering microbes for PET upcycling, prompting interest in their fundamental biochemical and structural elucidation. Terephthalate dioxygenase (TPADO) and its cognate reductase make up a complex multimetalloenzyme system that dihydroxylates TPA, activating it for enzymatic decarboxylation to yield protocatechuic acid (PCA). Here, we report structural, biochemical, and bioinformatic analyses of TPADO. Together, these data illustrate the remarkable adaptation of TPADO to the TPA dianion as its preferred substrate, with small, protonatable ring 2-carbon substituents being among the few permitted substrate modifications. TPADO is a Rieske [2Fe2S] and mononuclear nonheme iron-dependent oxygenase (Rieske oxygenase) that shares low sequence similarity with most structurally characterized members of its family. Structural data show an α-helix–associated histidine side chain that rotates into an Fe (II)–coordinating position following binding of the substrate into an adjacent pocket. TPA interactions with side chains in this pocket were not conserved in homologs with different substrate preferences. The binding mode of the less symmetric 2-hydroxy-TPA substrate, the observation that PCA is its oxygenation product, and the close relationship of the TPADO α-subunit to that of anthranilate dioxygenase allowed us to propose a structure-based model for product formation. Future efforts to identify, evolve, or engineer TPADO variants with desirable properties will be enabled by the results described here.