ZFIN ID: ZDB-PUB-070625-9
Replacement of Buried Cysteine from Zebrafish Cu/Zn Superoxide Dismutase and Enhancement of Its Stability via Site-Directed Mutagenesis
Ken, C.F., Lin, C.T., Wen, Y.D., and Wu, J.L.
Date: 2007
Source: Marine biotechnology (New York, N.Y.)   9(3): 335-342 (Journal)
Registered Authors: Wu, Jen-Leih
Keywords: Cu/Zn superoxide dismutase, I-Mutant 2.0, oxidative stress, site-direct mutagenesis, thermostability
MeSH Terms:
  • Amino Acid Sequence
  • Animals
  • Cysteine/genetics*
  • Enzyme Stability
  • Gene Expression Regulation, Enzymologic
  • Hot Temperature
  • Larva/drug effects
  • Mutagenesis, Site-Directed*
  • Paraquat/toxicity
  • Superoxide Dismutase/chemistry*
  • Superoxide Dismutase/genetics*
  • Zebrafish*/genetics
PubMed: 17549562 Full text @ Mar. Biotechnol.
ABSTRACT
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.
ADDITIONAL INFORMATION