Discovery and structural analysis of glycoside hydrolase family 176 α-1,2 glucosidase from Arthrobacter humicola A8F5.

Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process α-1,4- and α-1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as α-1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel α-1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves α-1,2-linkages via an anomer-inverting mechanism, with a preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 Å) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts an (α/α)6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses α-1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare α-glucans.