Structural Biology Research Center, IMSS, KEK* Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Japan** Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Japan***
○Masamichi Nagae* Atsuko Tsuchiya** Takane Katayama*** Kenji Yamamoto** Soichi Wakatsuki* Ryuichi Kato*
A novel 1,2-α-L-fucosidase (AfcA) from Bifidobacterium bifidum, which hydrolyzes the α1,2 glycosidic linkage of Fucα1-2Gal via an inverting mechanism, was isolated recently and classified as a member of a glycoside hydrolase family 95 (GH95). The catalytic activity of AfcA is ascribed to the middle of the polypeptide chain (Fuc domain). The enzymatic activity of the Fuc domain is strongly inhibited by deoxyfuconojirimycin (DFJ), which is an analogue of α-L-fucose. To elucidate the molecular mechanism of the enzyme, we report the X-ray crystal structures of AfcA Fuc domain in unliganded and complexed forms with DFJ, 2'fucosyllactose (substrate), and fucose and lactose (products) at 1.12-2.10 Å resolution. Overall structure of Fuc domain is composed of four regions: an N-terminal β region, a helical linker, a helical barrel region and a C-terminal β region. The overall domain arrangement is similar to those of maltose phosphorylase from Lactobacillus brevis (GH65) and chitobiose phosphorylase from Vibrio proteolyticus (GH95). The helical barrel region of Fuc domain shows resemblance to the (α/α)6 barrel structure of clan GH-L. In the complex structures, the ligands are deeply buried in the central cavity of the helical barrel region. From the crystal structures and mutational analyses, highly conserved Glu566 is strongly suggested to be the general acid catalyst. However, no carboxylic acid residue is located at the suitable position for the general base catalyst. We propose and discuss a possiblly new catalytic reaction mechanism of AfcA based on the structural and biochemical analyses.