Monothiol glutaredoxin cDNA from taiwanofungus camphorata: A novel CGFS-type glutaredoxin possessing glutathione reductase activity

Chi Tsai Lin, Chuian Fu Ken, I. Jing Chen, Chao Ting Lin, Shiu Mei Liu, Lisa Wen

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8 Citations (Scopus)

Abstract

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 (K M) 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 C 165→S165 lost 40-50% GR activity, whereas the mutant S168→C168 showed a 20% increase in its GR activity.

Original languageEnglish
Pages (from-to)3828-3835
Number of pages8
JournalJournal of Agricultural and Food Chemistry
Volume59
Issue number8
DOIs
Publication statusPublished - 2011 Apr 27

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All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Agricultural and Biological Sciences(all)

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