A Self-Cross-Linked Dual-Enzyme Assembly for Safe and Efficient Hyperuricemia Therapy.

Hyperuricemia, caused by purine metabolism disorders, is associated with gout, kidney stones, and cardiovascular disease. While urate oxidase (UOX) therapy reduces uric acid by converting it to allantoin, the generation of hydrogen peroxide (H₂O₂) limits its safety and efficacy due to oxidative stress. To overcome this limitation, we developed a modular enzyme assembly that colocalizes UOX with catalase (CAT), an H₂O₂-degrading enzyme, via engineered coiled-coil motifs. Complementary motifs bearing site-specific cysteine residues were designed to promote disulfide-stabilized heterodimerization, and their binding specificity and affinity were validated using a fluorescence-based assay. These motifs were then fused to UOX and CAT to enable spontaneous and stable assembly of a functional enzyme complex without chemical conjugation. The resulting complex exhibited high catalytic efficiency and significantly reduced reactive oxygen species-mediated cytotoxicity in human colon carcinoma cells. This work provides a tunable strategy for dual-enzyme assembly, enhancing therapeutic synergy while minimizing oxidative side effects in hyperuricemia treatment.