Rational engineering of Rieske dioxygenases for biodegradation of toxic aromatic compounds

The ever-growing pollution, persistence, and recalcitrance of toxic aromatic compounds require highly efficient biocatalytic solutions that outperform natural microbial metabolism. Rieske (2Fe-2S)-type dioxygenases (ROs) are a promising class of enzymes known to degrade various toxic aromatic compounds by hydroxylating aromatic rings, thereby facilitating their cleavage. ROs include biphenyl dioxygenase (BPDO), phthalate dioxygenase (PDO), terephthalate dioxygenase (TPDO), and isophthalate dioxygenase (IPDO), which degrade their respective substrates into the corresponding cis-dihydrodiols. Although they have narrow substrate-specificity constraints, they can be customized by redesigning the active-site architecture and substrate-access tunnels to generate variants with a broader substrate range and enhanced catalytic efficiency. Rational engineering of IPDO has demonstrated the advantage of introducing targeted mutations to expand substrate scope to include multiple phthalate regioisomers without compromising catalytic performance. Similarly, mutational studies of BPDO have shown that substituting interacting residues can increase conformational flexibility and accommodate structurally diverse substrates, such as polychlorinated biphenyls. This review analyzes the rational engineering of various Rieske dioxygenase enzymes, based on structural studies, to expand their catalytic range for the biodegradation of toxic aromatic pollutants.