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

Research output: Contribution to journalArticle

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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Taiwanofungus
Glutaredoxins
Glutathione Reductase
glutathione-disulfide reductase
Complementary DNA
Enzymes
enzymes
Antrodia
Taiwanofungus camphoratus
nitrilotriacetic acid
Nitrilotriacetic Acid
mutants
reducing agents
Glutathione Disulfide
heat inactivation
Reducing Agents
Mus musculus
Electrophoresis
NADP
NADP (coenzyme)

All Science Journal Classification (ASJC) codes

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

Cite this

@article{0f8cf6b64bea4733a4cd14967666aad2,
title = "Monothiol glutaredoxin cDNA from taiwanofungus camphorata: A novel CGFS-type glutaredoxin possessing glutathione reductase activity",
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.",
author = "Lin, {Chi Tsai} and Chuian-Fu Ken and Chen, {I. Jing} and Lin, {Chao Ting} and Liu, {Shiu Mei} and Lisa Wen",
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pages = "3828--3835",
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Monothiol glutaredoxin cDNA from taiwanofungus camphorata : A novel CGFS-type glutaredoxin possessing glutathione reductase activity. / Lin, Chi Tsai; Ken, Chuian-Fu; Chen, I. Jing; Lin, Chao Ting; Liu, Shiu Mei; Wen, Lisa.

In: Journal of Agricultural and Food Chemistry, Vol. 59, No. 8, 27.04.2011, p. 3828-3835.

Research output: Contribution to journalArticle

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T1 - Monothiol glutaredoxin cDNA from taiwanofungus camphorata

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