Contribution of a salt bridge triad to the thermostability of a highly alkaline protease from an alkaliphilic Bacillus strain

Crystallographic analysis of the highly alkaline M-protease from an alkaliphilic Bacillus strain shows the occurrence of a unique salt bridge triad Arg19-Glu271-Arg275 (in subtilisin BPN' numbering), which is not found in less alkaline true subtilisins BPN' and Carlsberg from Bacillus amyloliquefaciens and Bacillus licheniformis, respectively. Because the corresponding residues are all Gln residue in the subtilisin BPN', Gln residue was engineered into the position(s) 19, 271 and/or 275 in M-protease by site-directed mutagenesis. Disruptions of the salt bridge caused the reduction of the thermostability of the mutant proteins at alkaline pH with the following decreasing order of thermal inactivation rate; the wild-type > Arg275 -> Gln > Glu271 -> Gln > Arg19 -> Gln/Glu271 -> Gln/Arg275 -> Gln > Arg19 -> Gln. This result provides the evidence that the salt bridge triad contributes to the thermostability and structural rigidity of the highly alkaline M-protease.