Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. β-glucosidases (EC 3.2.1.21) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 β-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-β-d-glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-β-d-xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 Å resolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/β sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.

双歧杆菌是在人类肠道中定殖的益生菌的重要属。这些细菌可以摄取低聚糖，以进行己糖和戊糖的发酵代谢，产生乳酸，乙酸以及短链脂肪酸和丙酸酯。已知这些最终产品对人类健康具有重要影响。β-葡萄糖苷酶（EC 3.2.1.21）是双歧杆菌代谢和体内稳态的关键酶，因为它们水解小而可溶的糖类，通常会产生葡萄糖。在这里，我们描述了益生菌青春双歧杆菌GH3β-葡萄糖苷酶的克隆，表达，生化特性和第一个X射线结构（BaBgl3）。纯化的BaBgl3在45°C和pH 6.5下显示最大活性。在最佳条件下，BaBgl3是4-硝基苯基-β-高活性d吡喃葡萄糖苷（PNPG），并且在更小的程度，对4-硝基苯基β- d-木吡喃糖苷（pNPX，约占pNPG活性的32％）。BaBgl3的2.4Å分辨率晶体结构显示了一个由TIM桶形结构域组成的三结构域结构，该结构与α/β夹心结构域一起容纳了活性位点，并具有功能未知的第三C端纤连蛋白III型（FnIII）结构域。活性位点中底物的建模表明，天冬氨酸与pNPG中存在但pNPX中不存在的C6的羟基相互作用，这解释了底物的偏爱。最后，该酶被甘油和半乳糖显着稳定，导致酶活性及其寿命显着增加。