Ancestral sequence evolutionary trace and crystal structure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins.
The crystal structure of alkaline liquefying alpha-amylase (AmyK) from the alkaliphilic Bacillus sp. KSM-1378 was determined at 2.1 A resolution. The AmyK structure belongs to the GH13 glycoside hydrolase family, which consists of three domains, and bound three calcium and one sodium ions. The alkaline adaptation mechanism of AmyK was investigated by the ancestral sequence evolutionary trace method and by extensive comparisons between alkaline and nonalkaline enzyme structures, including three other protein families: protease, cellulase, and phosphoserine aminotransferase. The consensus change for the alkaline adaptation process was a decrease in the Lys content. The loss of a Lys residue is associated with ion pair remodeling, which mainly consists of the loss of Lys-Asp/Glu ion pairs and the acquisition of Arg ion pairs, preferably Arg-Glu. The predicted replacements of the positively charged amino acids were often, although not always, used for ion pair remodeling.