Biochemical and structural analyses of cooperative inhibition mechanism of 1,2-α-L-fucosidase (AfcA) by deoxyfuconojirimycin and lactose

Graduate School of Biostudies, Kyoto University* Structural Biology Research Center, Photon Factory, Insititute of Materials Structure Science, High Energy Accelerator Research Organization (KEK)** Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University***
â—‹Atsuko Tsuchiya* Masamichi Nagae** Soichi Wakatsuki** Ryuichi Kato** Takane Katayama*** Kenji Yamamoto*

Fucosidases have been used as a powerful tool to elucidate the function of α-L-fucosyl residues. Recently, we had cloned a gene encoding α-(1-2)-specific fucosidase (afcA), which comprises 1959 amino acids, from Bifidobacterium bifidum. We also showed that the middle of the polypeptide chain (Fuc domain) is responsible for the catalytic activity. We determined the crystal structure of Fuc domain of the apo form at 1.12 Å resolution and propose its catalytic mechanism.
To further investigate the biochemical aspects of Fuc domain, we searched for inhibitors of this enzyme and found that L-fucose or lactose weakly inhibits the fucosidase activity with a Ki value of mM range, and deoxyfuconojirimycin (DFJ) which is an analogue of α-L-fucose competitively inhibits the activity with a Ki value of 42 μM. We solved the crystal structure of the Fuc domain in complex with DFJ at 2.1 Å resolution. In the structure, DFJ bound to the putative catalytic pocket of the protein. Furthermore, we found that lactose synergistically inhibits the activity in the presence of DFJ. To elucidate the synergistic inhibition mechanism, we also determined the crystal structure of the Fuc domain in a ternary complex with DFJ and lactose. In this structure, the position of DFJ is identical to that of sole DFJ in the binary complex and the galactose moiety of lactose is deeply buried into the catalytic pocket, which prevents the release of DFJ from the protein. Based on these observations, we will discuss the inhibition mechanism of DFJ and the synergistic effect of lactose.