Evolution of alpha-amylases: Architectural features and key residues in the stabilization of the (beta/alpha)(8) scaffold

We provide a comprehensive analysis of the current enzymes with a-amylase a ctivity (AAMYs) that belong to family 13 glycoside hydrolase (GH-13; 144 Ar chaea, Bacteria, and Eukaryota sequences from 87 different species). This s tudy aims to further knowledge of the evolutionary molecular relationships among the sequences of their A and B domains with special emphasis on the c orrelation between what is observed in the structures and protein evolution . Multialignments for the A domain distinguish two clusters for sequences f rom Archaea organisms, eight for sequences from Bacteria organisms, and thr ee for sequences from Eukaryota organisms. The clusters for Bacteria do not follow any strict taxonomic pathway; in fact, they are rather scattered. W hen we compared the A domains of sequences belonging to different kingdoms, we found that various pairs of clusters were significantly similar. Using either sequence similarity with crystallized structures or secondary-struct ure prediction methods, we identified in all AAMYs the eight putative beta -strands that constitute the beta -sheet in the TIM barrel of the A domain and studied the packing in its interior. We also discovered a "hidden homol ogy" in the TIM barrel, an invariant Gly located upstream in the sequence b efore the conserved Asp in beta -strand 3. This Gly precedes an alpha -heli x and is actively involved in capping its N-terminal end with a capping box . In all cases, a Schellman motif caps the C-terminal end of this helix.