Structural similarities and evolutionary relationships in chloride-dependent α-amylases

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and,Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding chloride ion. All belong to animals, including mammals, birds, insects, acari, found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca2+ and Cl- binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dr:pendent alpha-amylases. (C) 2000 Elsevier Science B.V. All rights reserved.