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题名: Monothiol Glutaredoxin cDNA from Taiwanofungus Camphorata: A Novel CGFS-type Glutaredoxin Possessing Glutathione Reductase Activity
作者: Ken, Chuian-Fu;Chen, I-Jing;Lin, Chao-Ting;Liu, Shiu-Mei;Wen, Lisa;Lin, Chi-Tsai
贡献者: 生物學系
关键词: Taiwanofungus camphorata;Monothiol glutaredoxin;Three-dimensional (3-D) homology structure;Glutathione reductase
日期: 2011-01
上传时间: 2013-06-05T08:48:26Z
出版者: American Chemical Society
摘要: Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni2+-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (KM) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzyme's half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (kd) was 5.37 × 10−2 min−1. The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C40→S40 and C165→S165 lost 40−50% GR activity, whereas the mutant S168→C168 showed a 20% increase in its GR activity.
關聯: Journal of Agricultural and Food Chemistry, 59(8): 3828-3835
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