Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability.
A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.
Lin
Miao-Wen
MW
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 202.
Lin
Ming-Tse
MT
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002957:Citrus / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000201:enzymology*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013482:Superoxide Dismutase