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

The alpha-amylase sequences contained in databanks were screened for the pr esence of amino acid residues Arg195, Asn298 and,Arg/Lys337 forming the chl oride-binding site of several specialized alpha-amylases allosterically act ivated by this anion. This search provides 38 alpha-amylases potentially bi nding chloride ion. All belong to animals, including mammals, birds, insect s, acari, found in three extremophilic Gram-negative bacteria. An evolution ary distance tree based on complete amino acid sequences was constructed, r evealing four distinct clusters of species. On the basis of multiple sequen ce alignment and homology modeling, invariable structural elements were def ined, corresponding to the active site, the substrate binding site, the acc essory binding sites, the Ca2+ and Cl- binding sites, a protease-like catal ytic triad and disulfide bonds. The sequence variations within functional e lements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dr:pende nt alpha-amylases. (C) 2000 Elsevier Science B.V. All rights reserved.