Article

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

Authors: Yi Shi1, Ting Wang1,2,3, X. Edward Zhou4, Qiu-feng Liu1, Yi Jiang1,2, H. Eric Xu1,2,3,4
1 The CAS Key Laboratory of Receptor Research, VARI-SIMM Center, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
3 School of Life Science and Technology, ShanghaiTech University, Shanghai 201213, China
4 Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, MI 49503, USA
Correspondence to: Yi Jiang: shiyi@simm.ac.cn, H. Eric Xu: eric.xu@simm.ac.cn,
DOI: 10.1038/s41401-019-0269-x
Received: 24 April 2019
Accepted: 8 June 2019
Advance online: 28 June 2019

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.
Keywords: uricase; thermostability; disulfide cross-linking; crystal structure

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