Ishikawa Kazuhiko, Kataoka Misumi, Yanamoto Toshiaki, Nakabayashi Makoto, Watanabe Masahiro, Ishihara Satoru, Yamaguchi Shotaro
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-hiroshima, Japan.
Gifu R&D Center, Amano Enzyme Inc., Kakamigahara, Japan.
FEBS J. 2015 Jul;282(13):2540-52. doi: 10.1111/febs.13298. Epub 2015 May 7.
β-Galactosidase (EC 3.2.1.23) from Bacillus circulans ATCC 31382, designated BgaD, exhibits high transglycosylation activity to produce galacto-oligosaccharides. BgaD has been speculated to have a multiple domain architecture including a F5/8-type C domain or a discoidin domain in the C-terminal peptide region from amino acid sequence analysis. Here, we solved the first crystal structure of the C-terminal deletion mutant BgaD-D, consisting of sugar binding, Glyco_hydro, catalytic and bacterial Ig-like domains, at 2.5 Å. In the asymmetric unit, two molecules of BgaD-D were identified and the value of VM was estimated to be 5.0 Å(3) · Da(-1). It has been speculated that BgaD-D consists of four domains. From the structural analysis, however, we clarified that BgaD-D consists of five domains. We identified a new domain structure comprised of β-sheets in BgaD. The catalytic domain exhibits a TIM barrel structure with a small pocket suited for accommodating the disaccharides. Detailed structural information for the amino acid residues related to activity and substrate specificity was clarified in the catalytic domain. Furthermore, using the structural information, we successfully constructed some thermostable mutants via protein engineering method.
Coordinates for the BgaD-D structure have been deposited in the Protein Data Bank under accession code 4YPJ.
来自环状芽孢杆菌ATCC 31382的β-半乳糖苷酶（EC 3.2.1.23），命名为BgaD，具有高转糖基化活性以产生低聚半乳糖。通过氨基酸序列分析推测BgaD具有多结构域架构，包括在C端肽区域的一个F5/8型C结构域或一个盘状结构域。在此，我们解析了C端缺失突变体BgaD-D的首个晶体结构，其由糖结合、糖水解、催化和细菌Ig样结构域组成，分辨率为2.5 Å。在不对称单元中，鉴定出两个BgaD-D分子，VM值估计为5.0 Å(3)·Da(-1)。据推测BgaD-D由四个结构域组成。然而，通过结构分析，我们明确BgaD-D由五个结构域组成。我们在BgaD中鉴定出一种由β折叠组成的新结构域结构。催化结构域呈现出TIM桶状结构，带有一个适合容纳二糖的小口袋。在催化结构域中明确了与活性和底物特异性相关的氨基酸残基的详细结构信息。此外，利用这些结构信息，我们通过蛋白质工程方法成功构建了一些热稳定突变体。
BgaD-D结构的坐标已存入蛋白质数据库，登录号为4YPJ。
