Nakazawa Yutaka, Kageyama Masumi, Matsuzawa Tomohiko, Liang Ziqin, Kobayashi Kaito, Shimizu Hisaka, Maeda Kazuki, Masuhiro Miho, Motouchi Sei, Kumano Saika, Tanaka Nobukiyo, Kuramochi Kouji, Nakai Hiroyuki, Taguchi Hayao, Nakajima Masahiro
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa, 761-0795, Japan.
Commun Biol. 2025 Jan 16;8(1):66. doi: 10.1038/s42003-025-07494-1.
Galactosides are major carbohydrates that are found in plant cell walls and various prebiotic oligosaccharides. Studying the detailed biochemical functions of β-galactosidases in degrading these carbohydrates is important. In particular, identifying β-galactosidases with new substrate specificities could help in the production of potentially beneficial oligosaccharides. In this study, we identify a β-galactosidase with novel substrate specificity from Bacteroides xylanisolvens, an intestinal bacterium. The enzyme do not show hydrolytic activity toward natural β-galactosides during the first screening. However, when α-D-galactosyl fluoride (α-GalF) as a donor substrate and galactose or D-fucose as an acceptor substrate are incubated with a nucleophile mutant, reaction products are detected. The galactobiose produced from the α-GalF and galactose is identified as β-1,2-galactobiose using NMR. Kinetic analysis reveals that this enzyme effectively hydrolyzes β-1,2-galactobiose and β-1,2-galactotriose. In the complex structure with methyl β-galactopyranose as a ligand, the ligand is only located at subsite +1. The 2-hydroxy group and the anomeric methyl group of methyl β-galactopyranose faces in the direction of subsite -1 and the solvent, respectively. This observation is consistent with the substrate specificity of the enzyme regarding linkage position and chain length. Overall, we conclude that the enzyme is a β-galactosidase acting on β-1,2-galactooligosaccharides.
半乳糖苷是存在于植物细胞壁和各种益生元寡糖中的主要碳水化合物。研究β-半乳糖苷酶在降解这些碳水化合物中的详细生化功能很重要。特别是，鉴定具有新底物特异性的β-半乳糖苷酶有助于生产潜在有益的寡糖。在本研究中，我们从肠道细菌解木聚糖拟杆菌中鉴定出一种具有新型底物特异性的β-半乳糖苷酶。在首次筛选中，该酶对天然β-半乳糖苷不显示水解活性。然而，当将α-D-半乳糖基氟化物（α-GalF）作为供体底物，半乳糖或D-岩藻糖作为受体底物与亲核突变体一起孵育时，可检测到反应产物。使用核磁共振将由α-GalF和半乳糖产生的半乳糖二糖鉴定为β-1,2-半乳糖二糖。动力学分析表明，该酶能有效水解β-1,2-半乳糖二糖和β-1,2-半乳糖三糖。在以β-吡喃半乳糖甲酯作为配体的复合物结构中，该配体仅位于亚位点+1。β-吡喃半乳糖甲酯的2-羟基和异头甲基分别朝向亚位点-1和溶剂的方向。这一观察结果与该酶在连接位置和链长方面的底物特异性一致。总体而言，我们得出结论，该酶是一种作用于β-1,2-半乳糖寡糖的β-半乳糖苷酶。
