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

Several bacteria possess components of catabolic pathways for the synthetic polyesterpoly(ethylene terephthalate) (PET). These proceed by hydrolyzing the ester linkages ofthe polymer to its monomers, ethylene glycol and terephthalate (TPA), which are fur-ther converted into common metabolites. These pathways are crucial for geneticallyengineering microbes for PET upcycling, prompting interest in their fundamental bio-chemical and structural elucidation. Terephthalate dioxygenase (TPADO) and its cog-nate reductase make up a complex multimetalloenzyme system that dihydroxylatesTPA, 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 dia-nion as its preferred substrate, with small, protonatable ring 2-carbon substituentsbeing among the few permitted substrate modifications. TPADO is a Rieske [2Fe2S]and mononuclear nonheme iron-dependent oxygenase (Rieske oxygenase) that shareslow sequence similarity with most structurally characterized members of its family.Structural data show anα-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 withdifferent substrate preferences. The binding mode of the less symmetric 2-hydroxy-TPA substrate, the observation that PCA is its oxygenation product, and the close rela-tionship of the TPADOα-subunit to that of anthranilate dioxygenase allowed us topropose a structure-based model for product formation. Future efforts to identify,evolve, or engineer TPADO variants with desirable properties will be enabled by theresults described here.