Structure and Function of Honeybee α-Amylase of Glycoside Hydrolase Family 13 Subfamily 15.

α-Amylase, ubiquitously distributed across diverse organisms, catalyzes the hydrolysis of the internal α-(1→4)-linkage of α-(1→4)-glucan including starch as an essential energy source. Insect α-amylases, which belong to the glycoside hydrolase family 13 subfamily 15 (GH13_15), are important for optimal larval growth and adult longevity. Honeybee (Apis mellifera) α-amylase (AMA) is expressed in the hypopharyngeal glands of forager bees and secreted into honey. AMA in honey, which is important for food quality control, has been partly characterized. However, its structure-function relationship is poorly understood. Herein, we present biochemical, structural, and mutational analyses of AMA. Kinetic analysis using p-nitrophenyl maltooligosaccharides and their 4,6-benzylidene-modified derivatives revealed a subsite affinity map of AMA. AMA contains high-affinity subsites -3, -2, +1, and +2, similar to those of the mammalian α-amylases of GH13_24. Most AMA substrate-binding residues are conserved in the GH13_24 enzymes. Mutational analysis revealed that Leu175 is crucial at subsites -3/-2 for reactions with oligo- and polysaccharides. Furthermore, Trp77 at subsites -3/-2 and Lys210 at subsite +2 are suggested to be involved in the proper binding of long-chain substrates. AMA shares a surface sugar-binding site with mammalian α-amylases, where Asn281, Trp285, and Trp293 are essential for the binding and degradation of starch granules.