title: Characterization and structural analysis of human selenium-dependent glutathione peroxidase 4 mutant expressed in Escherichia coli.
abstract: Glutathione peroxidase 4 (GPx4) is a monomeric selenium-dependent glutathione peroxidase highly expressed in mammalian cells, which can reduce phospholipid hydroperoxides. However, it has been difficult to express recombinant mammalian GPx4 in Escherichia coli because of the differences in the selenocysteine (Sec) incorporation machinery between eukaryotes and prokaryotes. In this study, an E. coli BL21(DE3)cys auxotrophic strain was used to express GPx4 mutants. We found that untargeted substitution of Cys-2, Cys-37, Cys-75, Cys-107, and Cys-148 with Sec led to loss of activity, suggesting that mutation of any of these Cys residues in GPx4 could result in a structural change. Additionally, we found that the catalytic activity of GPx4 mutants increased as the number of noncatalytic Sec residues decreased, indicating that the negative effects could be mitigated by replacing these Cys residues with Ser residues. A GPx4 mutant with all Cys residues converted to Ser exhibited a "Ping-Pong" mechanism and structure similar to that of native GPx4, indicating that it could act as a substitute for GPx4, when heterologously expressing the protein in E. coli. This research provides an important foundation for biosynthesis of selenium-dependent GPx4 mutants in E. coli.
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: College of Electronic Science and Engineering, Changchun 130000, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Song
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Guo
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, China.
authors.forename: Shuan
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, China.
authors.forename: Long
authors.initials: L
authors.lastname: Chen
authors.affiliation: College of Pharmaceutical Science, Jilin University, Changchun 130021, China; State Key Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun 130000, China. Electronic address: jingyanweijluedu@163.com.
authors.forename: Jingyan
authors.initials: J
authors.lastname: Wei
mesh_terms: D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005979:Glutathione Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D010957:Plasmids; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017279:Selenocysteine / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000737:chemistry* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D017279:Selenocysteine; D012694:Serine; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D003545:Cysteine
keywords: Antioxidants; Cys auxotrophic strain; Free radicals; Glutathione peroxidase 4; Mutants; Overexpression; Selenoenzymes