Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from Taiwanofungus camphorata.

Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) 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+)-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of approximately 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 (K(m)) values for beta-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 degrees C was 8.5 min, and its thermal inactivation rate constant K(d) 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.