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)