Abstract
Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU 193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed Into Escherichia coli. Functional TcGrx was expressed and purified by Ni 2+-nitrllotriacetic acid Sepharose. The purified enzyme showed bands of ∼15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (Km) values for β-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 °C was 8.5 min, and its thermal inactivation rate constant Kd was 6.52 x 10-2 min-1. The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.
| Original language | English |
|---|---|
| Pages (from-to) | 10357-10362 |
| Number of pages | 6 |
| Journal | Journal of Agricultural and Food Chemistry |
| Volume | 57 |
| Issue number | 21 |
| DOIs | |
| Publication status | Published - 2009 Nov 11 |
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All Science Journal Classification (ASJC) codes
- Chemistry(all)
- Agricultural and Biological Sciences(all)
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Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from taiwanofungus camphorata. / Ken, Chuian Fu; Lin, Choa Yi; Jiang, Yu Chi; Wen, Lisa; Lin, Chi Tsai.
In: Journal of Agricultural and Food Chemistry, Vol. 57, No. 21, 11.11.2009, p. 10357-10362.Research output: Contribution to journal › Article
TY - JOUR
T1 - Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from taiwanofungus camphorata
AU - Ken, Chuian Fu
AU - Lin, Choa Yi
AU - Jiang, Yu Chi
AU - Wen, Lisa
AU - Lin, Chi Tsai
PY - 2009/11/11
Y1 - 2009/11/11
N2 - Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU 193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed Into Escherichia coli. Functional TcGrx was expressed and purified by Ni 2+-nitrllotriacetic acid Sepharose. The purified enzyme showed bands of ∼15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (Km) values for β-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 °C was 8.5 min, and its thermal inactivation rate constant Kd was 6.52 x 10-2 min-1. The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.
AB - Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU 193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed Into Escherichia coli. Functional TcGrx was expressed and purified by Ni 2+-nitrllotriacetic acid Sepharose. The purified enzyme showed bands of ∼15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (Km) values for β-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 °C was 8.5 min, and its thermal inactivation rate constant Kd was 6.52 x 10-2 min-1. The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.
UR - http://www.scopus.com/inward/record.url?scp=70449101120&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=70449101120&partnerID=8YFLogxK
U2 - 10.1021/jf9021256
DO - 10.1021/jf9021256
M3 - Article
C2 - 19886686
AN - SCOPUS:70449101120
VL - 57
SP - 10357
EP - 10362
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
SN - 0021-8561
IS - 21
ER -