Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis.
Zebrafish Cu/Zn-superoxide dismutase (ZSOD1) has one free cysteine (Cys-7) in a first beta-strand with lower thermostability. We predicted the stability would be increased with single-point mutation at 70 degrees C via the I-Mutant 2.0 server, and generated a mutant SOD with replacement of the free Cys to Ala (ZSODC7A) by site-directed mutagenesis. The mutant was expressed and purified from the Escherichia coli strain AD494(DE3)pLysS and the yield was 2 mg from 0.4 L of culture. The ZSODC7A was heated at 90 degrees C. In a time-dependent assay, the time interval for 50% inactivation was 32 min, and its thermal inactivation rate constant K (d) was 2 x 10(-2) min(-1). The mutant was still activated in broad pH range (2.3-12), and had only a moderate effect under sodium dodecyl sulfate treatment. The calculated specific activity of the mutant was 3980 U/mg, twice that of wild-type ZSOD1. In addition, we soaked fish larva with equal enzyme units of either ZSOD1 or ZSODC7A for 2 h, and then stressed them with 100 ppm of paraquat to induce oxidative injury. The survival rate was significant.
Ken
Chuian-Fu
CF
Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan.
Lin
Chi-Tsai
CT
Wen
Yu-Der
YD
Wu
Jen-Leih
JL
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics*; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D006358:Hot Temperature; D007814:Larva / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D010269:Paraquat / Q000633:toxicity; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics
D013482:Superoxide Dismutase; D003545:Cysteine; D010269:Paraquat