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

Area of Science:

• Biochemistry and Molecular Biology
• Environmental Microbiology
• Biocatalysis and Enzyme Engineering

Background:

• Phthalates are persistent environmental pollutants with significant ecological and health impacts.
• Microbial degradation offers a sustainable route for phthalate remediation.
• Isophthalate dioxygenase (IPDO) initiates isophthalate degradation, but its structure and substrate specificity are poorly understood.

Purpose of the Study:

• To determine the crystal structures of substrate-free IPDO and its complex with isophthalate.
• To elucidate the structural basis of IPDO's substrate specificity.
• To engineer IPDO variants with broadened substrate specificity for enhanced phthalate degradation.

Main Methods:

• X-ray crystallography was used to determine the structures of IPDO.
• Comparative structural analysis identified key residues influencing substrate binding.
• Site-directed mutagenesis was employed to engineer IPDO variants with altered substrate specificity.

Main Results:

• The first crystal structures of IPDO revealed a unique trimeric architecture (α3) distinct from related dioxygenases.
• Structural analysis identified steric and electrostatic constraints, particularly residue V178, limiting substrate scope.
• Engineered IPDO variants (V178A and F249H) demonstrated degradation of phthalate, isophthalate, and terephthalate with comparable catalytic efficiency.

Conclusions:

• This work provides critical structural insights into isophthalate dioxygenase's molecular mechanisms and substrate specificity.
• Rational engineering of IPDO successfully expanded its substrate range to include multiple phthalate regioisomers.
• The engineered IPDO variants represent promising biocatalysts for environmental detoxification and biotechnological applications.