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.
Ken
Chuian-Fu
CF
Institute of Biotechnology, National Changhua University of Education, Changhua 500, Taiwan.
Lin
Choa-Yi
CY
Jiang
Yu-Chi
YC
Wen
Lisa
L
Lin
Chi-Tsai
CT
D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D054477:Glutaredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020072:Polyporales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational
D005656:Fungal Proteins; D054477:Glutaredoxins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate)