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

查看全文 作　　者：Yi [1]Shi;Ting [1,2,3]Wang;X.Edward [4]Zhou;Qiu-feng [1]Liu;Yi [1,2]Jiang;H.Eric [1,2,3,4]Xu

机构地区：[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,Ml 49503,USA

出　　处：《Acta Pharmacologica Sinica》索引2019年第40卷第10期,共9页

基　　金：This work was supported by the National Natural Science Foundation of China(31770796 to YJ);the Strategic Priority Research Program of CAS(XDB08020303 to HEX);the National Science and Technology Major Project(2018ZX09711002-002-002 to YJ);the K.C.Wong Education Foundation(to YJ). 摘　　要：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,w e 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 WTAgUricase.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. 关键词：URICASE thermostability DISULFIDE cross-linking CRYSTAL structure

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

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