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Arthrobacter globiformis Uricase (AgUricase) is a homotetrameric uricase with the potential for therapeutic use in treating hyperuricemia-related diseases.To achieve sufficient therapeutic effects,it is essential for this enzyme to have high thermostability and long half-life in physiological condition.To improve the thermostability of this enzyme,we introduced a series of cysteine pair mutations into the AgUricase subunits based on its structural model and studied the thermostability of the mutant enzymes with introduced disulfide bridges.Two intersubunit cysteine pair mutations,K12C-E286C and S296C-S296C,were found to markedly increase the melting temperatures of the corresponding mutant enzymes compared with WT AgUricase.The crystal structure of the K12C-E286C mutant at 1.99 (A) resolution confirmed the formation of a distinct disulfide bond between the two subunits in the dimer.Structural analysis and biochemical data revealed that the C-terminal loop of AgUricase was flexible,and its interaction with neighboring subunits was required for the stability of the enzyme.We introduced an additional intersubunit K244C-C302 disulfide bond based on the crystal structure of the K12C-E286C mutant and confirmed that this additional disulfide bond further stabilized the flexible C-terminal loop and improved the thermostability of the enzyme.Disulfide cross-linking also protected AgUricase from protease digestion.Our studies suggest that the introduction of disulfide bonds into proteins is a potential strategy for enhancing the thermostability of multimeric proteins for medical applications.