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Structure-based design of a hyperthermostable AgUricase for hyperuricemia and gout therapy

	author = {Yi Shi and Ting Wang and X. Edward Zhou and Qiu-feng Liu and Yi Jiang and H. Eric Xu},
	title = {Structure-based design of a hyperthermostable AgUricase for hyperuricemia and gout therapy},
	journal = {Acta Pharmacologica Sinica},
	volume = {40},
	number = {10},
	year = {2019},
	keywords = {},
	abstract = {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 Å 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.},
	issn = {1745-7254},	url = {}