Cloning, Expression, and Purification of a Functional Glutathione Reductase from Sweet Potato (Ipomoea batatas [L.] Lam): Kinetic Studies and Characterization

摘要

A cDNA encoding a putative glutathione reductase (GR) was cloned from sweet potato (lb). 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 Za~(2+), but not by Ca~(2+). The protein's half-life of deactivation at 70 °C was 3.3min, and its thermal inactivation rate constant K_d was 3.48 x 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.