Cloning, Expression, and Characterization of a Thioredoxin Reductase cDNA from Taiwanofungus camphorata
2010
Huang, Zhiyu | Ken, Chuian-Fu | Chi, Hsiang-Hui | Wen, Lisa | Lin, Chi-Tsai
A cDNA encoding putative thioredoxin reductase (TR) was identified from a medicinal mushroom, Taiwanofungus camphorata (T. camphorata). Alignment of the deduced amino acid sequence with TRs from other organisms showed high levels of identity (59−74%). A three-dimensional (3-D) homology structure was created for this TR. Functional T. camphorata TR (TcTR) was overexpressed in yeast and purified. The purified enzyme showed a monomic form on a 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). The enzyme’s half-life of deactivation at 60 °C was 12.9 min, and its thermal inactivation rate constant Kd was 5.37 × 10(−2) min−1. The optimal pH for the enzyme was pH 8 and retained about 76% activity in the presence of 0.1 M imidazole. The enzyme showed 50% activity after 10 min of incubation at 37 °C with chymotrypsin. The Michaelis constant (Km) value for dithionitrobenzoate (DTNB) was 1.59 mM.
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