Crystal Structure of Truncated Fibrobacter succinogenes 1,3-1,4-β-d-Glucanase in Complex with β-1,3-1,4-Cellotriose

Fibrobacter succinogenes 1,3-1,4-β-d-glucanase (Fsβ-glucanase) catalyzes the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in β-d-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsβ-glucanase (TFsβ-glucanase) in complex with β-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 Å, reveal that the overall fold of TFsβ-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The β-1,3-1,4-cellotriose with subsites −3 to −1 bound to the active cleft of TFsβ-glucanase with its reducing end subsite −1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the β-1,3-1,4-cellotriose adopted a relaxed image conformation, with a β-1,3 glycosidic linkage between subsites −2 and −1, and a β-1,4 glycosidic linkage between subsites −3 and −2. On the basis of the enzyme–product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsβ-glucanase is proposed.