Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges.

An alkaline, mesophilic endo-1,4-beta-glucanase from alkaliphilic Bacillus sp. strain KSM-64 was significantly thermostabilized by replacement of both Asn179 and Asp194 with lysine by site-directed mutagenesis. Structural remodeling of the mutant enzyme newly generated by the double mutation suggested that Glu175-->Lys179 and Glu190-->Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the alpha(4)-helix from Glu175 to Ser195. By molecular dynamics simulations, the N(zeta) hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O(varepsilon1) and O(varepsilon2) of Glu175 and the carbonyl O(varepsilon1) of Glu190, respectively, with distances of around 2 A for all. These results confirm that the formation of these double intrahelical ion pairs (salt bridges) is responsible for the thermostabilization by the double mutation.