title: Unglycosylated recombinant human glutathione peroxidase 3 mutant from Escherichia coli is active as a monomer.
abstract: Glutathione peroxidase 3 (GPx3) is a glycosylated member of GPx family and can catalyze the reaction of different types of peroxides with GSH to form their corresponding alcohols in vitro. The active center of GPx3 is selenocysteine (Sec), which is incorporated into proteins by a specific mechanism. In this study, we prepared a recombinant human GPx3 (rhGPx3) mutant with all Cys changed to Ser from a Cys auxotrophic strain of E. coli, BL21(DE3)cys. Although lacking post-translational modification, rhGPx3 mutant still retained the ability to reduce H2O2 and PLPC-OOH. Study on the quaternary structure suggested that rhGPx3 mutant existed as a monomer in solution, which is different from native tetrameric GPx3. Loss of the catalytic activity was considered to be attributed to both the absence of glycosylation and the failure of the tetramer. Further analysis was performed to compare the structures of rhGPx3 and GPx4 mutant, which were quite similar except for oligomerization loop. The differences of amino acid composition and electrostatic potentials on the oligomerization loop may affect the binding of large substrates to rhGPx3 mutant. This research provides an important foundation for biosynthesis of functionally selenium-containing GPx3 mutant in E.coli.
authors.affiliation: College of Electronic Science and Engineering, Jilin University, 2699 Qianjin Street, Changchun 130012, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Song
authors.affiliation: College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun 130021, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: College of Electronic Science and Engineering, Jilin University, 2699 Qianjin Street, Changchun 130012, China.
authors.forename: Ruiqing
authors.initials: R
authors.lastname: Xing
authors.affiliation: College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun 130021, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Guo
authors.affiliation: College of Electronic Science and Engineering, Jilin University, 2699 Qianjin Street, Changchun 130012, China.
authors.forename: Dali
authors.initials: D
authors.lastname: Liu
authors.affiliation: 1] College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun 130021, China [2] State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130021, China.
authors.forename: Jingyan
authors.initials: J
authors.lastname: Wei
authors.affiliation: College of Electronic Science and Engineering, Jilin University, 2699 Qianjin Street, Changchun 130012, China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Song
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli; D005979:Glutathione Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D010545:Peroxides / Q000378:metabolism; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017279:Selenocysteine / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D010545:Peroxides; D017279:Selenocysteine; C486484:GPX3 protein, human; D005979:Glutathione Peroxidase