Résumé
Background: The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for
Bacillus licheniformis
α-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity.
Results: We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all
α-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder→order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in
α-amylases, is located at the interface between domains A and C.
Conclusions: We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder→order transition observed in BLA leads to the formation of the extended substrate binding site and explains on a structural level the calcium dependency of
α-amylases. Sequence comparisons indicate that the unique Ca–Na–Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial
α-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.