Activation of Bacillus licheniformis α-amylase through a disorder→order transition of the substrate-binding site mediated by a calcium–sodium–calcium metal triad

• Published in: Structure (London) Vol. 6; no. 3; pp. 281 - 292
• Main Authors: Machius, Mischa, Declerck, Nathalie, Huber, Robert, Wiegand, Georg
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.1998; Elsevier (Cell Press)
• Subjects: alpha-Amylases - chemistry; alpha-Amylases - metabolism; Bacillus - enzymology; Binding Sites; calcium; Calcium - chemistry; Calcium - metabolism; Crystallography, X-Ray; Enzyme Stability; Life Sciences; Models, Molecular; order→disorder transition; Protein Conformation; Protein Folding; sodium; Sodium - chemistry; Sodium - metabolism; thermostability; α-amylase; sodium; α-amylase; calcium; order→disorder transition; thermostability; BIOTECHNOLOGIE
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/S0969-2126(98)00032-X

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.