Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: thermodynamic studies and structural interpretation.

Abstract

This paper provides further understanding of the thermodynamic and structural features determining the stability of Bacillus licheniformis alpha-amylase (BLA) at two crucial positions, His133 and Ala209. Results of protein modelling and saturated site-directed mutagenesis at position 133 and 209 have been reported in a previous paper (Declerck et al., 1995, Prot. Engng, 8, 1029-1037). In the first part of the present work, evidence is presented supporting the hypothesis that the stabilizing mutations reduce the rate of initial unfolding of the enzyme during the reversible step of the inactivation reaction and do not modify the irreversible processes undergone subsequently by the unfolded molecules. In the second part, we have examined the three-dimensional structure of BLA which has been determined recently by X-ray analysis (Machius et al., 1995, J. Mol. Biol., 246, 545-559). This analysis showed that our previous predictions made from molecular modelling were partly correct. At position 209, the effect of the stabilizing substitutions can be explained by a groove-filling effect reinforcing the hydrophobic packing between two helices of the central domain, while preserving a well-ordered water structure at the surface. At position 133, the stabilizing substitutions must compensate the loss of the hydrogen bond network in which the original histidine side-chain is involved; this compensation could be achieved through enhanced hydrophobic side-chain interactions within the beta-sheet where residue 133 is located, which correlates with the propensity of the residue to form and maintain a beta-strand conformation of the main chain at this position.