Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: Understanding the molecular basis of protein thermostability

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify a mino acid residues essential for the thermostability of the SOD from Aquife x pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T-1/2 value of K12A was 210 min and that of the wild type was 175 min at 95 degre esC. However, the thermostability of the mutant E41A, which has a T-1/2 val ue of 25 min at 95 degreesC, was significantly decreased compared to the wi ld type of ApSOD. To explain the enhanced thermostability of K12A and therm olabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general s ignificance of hydrogen bonds and ion-pair network in the thermostability o f proteins. (C) 2001 Academic Press