Characterization and Structural Analysis of a Novel <i>exo</i>-Type Enzyme Acting on β‑1,2-Glucooligosaccharides from <i>Parabacteroides distasonis</i>

β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified <i>endo</i>-β-1,2-glucanase from <i>Chitinophaga pinensis</i> (<i>Cp</i>SGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a <i>Cp</i>SGL homologue from <i>Parabacteroides distasonis</i>, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading enzymes. BDI_3064 hydrolyzed linear β-1,2-glucan and β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of ≥4 to produce sophorose specifically but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new <i>exo</i>-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosaccharide analogues that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the nonreducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike <i>Cp</i>SGL. Superimposition of BDI_3064 and <i>Cp</i>SGL complexed with ligands showed that R93 in domain 1 overlapped subsite −3 in <i>Cp</i>SGL. Docking analysis involving a β-1,2-glucooligosaccharide with DP4 showed that R93 completely blocks the nonreducing end of the docked β-1,2-glucooligosaccharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the nonreducing end of substrates to act as an <i>exo</i>-type enzyme. Thus, we propose 2-β-d-glucooligosaccharide sophorohydrolase (nonreducing end) as a systematic name for BDI_3064