Protein Engineering, Vol. 14, No. 7, 501-504,
July 2001
© 2001 Oxford University Press
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
1 Tochigi Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497 and 2 Department of Biotechnology and Biomaterial Chemistry, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan
An alkaline, mesophilic endo-1,4-ß-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 Glu190Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the 
4-helix from Glu175 to Ser195. By molecular dynamics simulations, the N
hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O
1 and O2 of Glu175 and the carbonyl O1 of Glu190, respectively, with distances of around 2 Å 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.