The thermostable endo-1,5-α-L-arabinanase from Bacillus thermodenitrificans TS-3 (ABN-TS) hydrolyzes the α-1,5-L-arabinofuranoside linkages of arabinan. In this study, the crystal structures of inactive ABN-TS mutants, D27A and D147N, were determined in complex with arabino-oligosaccharides. The crystal structures revealed that ABN-TS has at least six subsites in the deep V-shaped cleft formed across one face of the propeller structure. The structural features indicate that substrate recognition is profoundly influenced by the remote subsites as well as by the subsites surrounding the active center. The `open' structure of the substrate-binding cleft of the endo-acting ABN-TS is suitable for the random binding of several sugar units in polymeric substrates.
Keywords: Bacillus thermodenitrificans; arabinanases; biofuels; crystal structure; endo-1,5-α-l-arabinanase mutants; endo-acting enzymes; enzyme–substrate complex; glycoside hydrolase family 43.