Structure-guided engineering of an aromatic ring-hydroxylating dioxygenase for broad-spectrum phthalate degradation

Phthalates such as isophthalate, phthalate, and terephthalate are widespread environmental pollutants with significant health and ecological impacts. <i>Comamonas testosteroni</i> KF1 initiates isophthalate degradation through a specialized two-component enzyme system composed of isophthalate dioxygenase (IPDO) and its cognate reductase, isophthalate dioxygenase reductase. Despite its environmental significance, the lack of structural insights into IPDO has hindered efforts to rationally redesign, optimize, and harness its chemistry. Here, we report the first crystal structures of substrate-free IPDO and its complex with isophthalate, revealing unique structural features that underpin its substrate specificity. Unlike related oxygenases, phthalate dioxygenase (α₃α₃) and terephthalate dioxygenase (α₃β₃), IPDO adopts a trimer (α₃) architecture, with distinct active site residues tailored to isophthalate binding. The comparative structural analysis identified steric and electrostatic constraints-particularly involving residue V178-that preclude the binding of ortho- or para-substituted substrates. Leveraging these structural insights, we engineered IPDO variants with broadened substrate specificity. Notably, the V178A and F249H substitutions enabled the enzyme to degrade three regioisomers of phthalate (phthalate, isophthalate, and terephthalate) without diminishing its native activity against isophthalate. The catalytic turnover (<i>k</i>_cat) of the V178A/F249H double mutant was found to be 4.8 ± 0.3, 4.9 ± 0.2, and 4.0 ± 0.2 s^-1 for isophthalate, terephthalate, and phthalate, respectively, demonstrating comparable catalytic efficiency for all three substrates. Overall, this work advances our understanding of the molecular mechanisms involved in isophthalate dihydroxylation and elucidates a rational engineering approach to expand the catalytic repertoire of IPDO for biotechnological and environmental applications.IMPORTANCEPhthalate pollution poses a major environmental concern due to its widespread use as plasticizers and its persistence in ecosystems. Microbial degradation of phthalates offers a sustainable solution for mitigating this contamination. Among the key enzymes involved, aromatic-ring-hydroxylating dioxygenases initiate the first critical step in phthalate breakdown. However, most known enzymes exhibit narrow substrate specificity, limiting their utility for degrading diverse phthalate isomers such as isophthalate, phthalate, and terephthalate. This research addresses a critical gap by elucidating the structural basis of substrate specificity in isophthalate dioxygenase and applying rational engineering to expand its catalytic range. By generating enzyme variants capable of degrading all three phthalate regioisomers, this work provides a blueprint for designing versatile biocatalysts tailored for pollutant detoxification.