Biochemical and structural characterization of AmyY from Alkalimonas sp. NCh-2 reveals a class of metal-free α-amylases in glycoside hydrolase family 13

α-Amylases (EC 3.2.1.1) are among the most important industrial enzymes in starch saccharification, detergent, paper, and textile industries. Canonical α-amylases typically contain Ca ²⁺ in their structure, which is not directly involved in catalysis but essential in maintaining the structural integrity of α-amylases. Ca ²⁺ ions are easily removed by chelating reagents, which may affect the activity and stability of α-amylases and thereby hinder their applications in some industries. Here, we identified a metal-free α-amylase, AmyY, from the bacterium Alkalimonas sp. NCh-2 isolated from an alkaline hot spring. AmyY contains a catalytic module of glycoside hydrolase family 13 (GH13) and a carbohydrate-binding module of family 20 (CBM20). AmyY exhibits high activity under alkaline conditions (pH 8.0–11.0) and tolerance to high salinity, chelating reagents, and surfactants. We solved the structures of AmyY and its complex with acarbose. Although the overall topology and active sites of AmyY resemble those of reported GH13 α-amylases, the structure of AmyY is devoid of metal ions. The inability of AmyY to bind metal ions results from replacements of acidic amino acid residues by neutral ones. Combined with biochemical, structural, and bioinformatic data, we demonstrated that AmyY and its homologs represent a class of metal-free α-amylases in GH13. Moreover, structural analyses revealed that the CBM20 of AmyY is highly flexible, and we obtained a mutant with enhanced thermostability by truncating the CBM20. This mutant demonstrates remarkable wash performance and desizing capability at alkaline pH, which underscores its application potential in the detergent and textile industries.