Cloning, expression, and purification of a functional glutathione reductase from sweet potato (Ipomoea batatas [L.] Lam): kinetic studies and characterization.
A cDNA encoding a putative glutathione reductase (GR) was cloned from sweet potato (Ib). The deduced protein showed high level of sequence homology with GRs from other plants (79-38%). A three-dimensional (3-D) homology structure was created. The active site Cys residues are conserved in all reported GR. Functional IbGR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 10% native polyacrylamide gel electrophoresis (PAGE). The monomeric nature of the enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular mass determination of the native enzyme. The Michaelis constant (K(m)) values for GSSG (glutathione disulfide) and NADPH (β-nicotinamide adenine dinucleotide phosphate, reduced form) were 0.114 and 0.056 mM, respectively. The enzyme activity was inhibited by Cu(2+) and Zn(2+), but not by Ca(2+). The protein's half-life of deactivation at 70 °C was 3.3 min, and its thermal inactivation rate constant K(d) was 3.48 × 10(-1) min(-1). The enzyme was active in a broad pH range from 6.0 to 11.0 and in the presence of imidazole up to 0.8 M. The native enzyme appeared to be resistant to digestion by trypsin or chymotrypsin.
Institute of Bioscience and Biotechnology and Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan.
Cheng-Jen
CJ
Chen
Chih-Yu
CY
Huang
Jenq-Kuen
JK
Huang
Choa-Yi
CY
Lin
Chi-Tsai
CT
Lin
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D027723:Ipomoea batatas / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D035281:Plant Tubers / Q000201:enzymology; D011994:Recombinant Proteins; D016415:Sequence Alignment
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D005980:Glutathione Reductase