α-Galactosidase/Sucrose Kinase (AgaSK), a Novel Bifunctional Enzyme from the Human Microbiome Coupling Galactosidase and Kinase Activities
α-Galactosidase/Sucrose Kinase (AgaSK), a Novel Bifunctional Enzyme from the Human Microbiome Coupling Galactosidase and Kinase Activities
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DOI：
10.1074/jbc.m111.286039
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发表时间：
2011-11-25
影响因子：
4.8
通讯作者：
Giardina, Thierry
中科院分区：
文献类型：
--
作者：
Bruel, Laetitia;Sulzenbacher, Gerlind;Giardina, Thierry
α-Cialactoside是一种广泛存在于植物中的不可消化碳水化合物。它们是我们日常食物中潜在的能量来源，它们被微生物区系同化可能在肥胖中发挥作用。在肠道中，它们被微生物糖苷酶降解，这些糖苷酶通常是带有与碳水化合物结合模块相连的催化域的模块化酶。在这里，我们介绍了一种来自人类肠道细菌瘤胃球菌E1的双功能酶，即α-半乳糖苷酶/蔗糖激酶(AgaSK)。序列分析表明，AgaSK由两个结构域组成：一个与糖苷水解酶家族GH36的α-半乳糖苷酶密切相关，另一个含有核苷酸结合基序。其生化特性表明，AgaSK能将蜂蜜二糖和棉子糖分别水解为半乳糖和葡萄糖或蔗糖，并在三磷酸腺苷存在下，将蔗糖特异性地磷酸化到葡萄糖的C6位。从棉子糖直接产生蔗糖-6-P指向细菌中的糖酵解途径，到目前为止还没有描述。半乳糖苷酶结构域在载脂蛋白形式和与产物的复合体中的晶体结构揭示了反应和底物识别机制，突出了有效底物结合所必需的寡聚状态，并暗示了半乳糖和激酶域之间的相互作用。
alpha-Cialactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, alpha-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to alpha-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically p hosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligorneric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.