Macromolecular Crystallography and Structural Biology Department, Institute of Physical-Chemistry Rocasolano, CSIC, Serrano 119, 28006, Madrid, Spain.
Institute of Catalysis and Petrochemistry, CSIC, Marie Curie 2, 28049, Madrid, Spain.
Sci Rep. 2019 Nov 25;9(1):17441. doi: 10.1038/s41598-019-53948-y.
Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). Only hydroquinone and hydroxytyrosol gave rise to the formation of glycosylated products. For the rest, an inhibitory effect on both the hydrolytic (H) and transglycosylation (T) activity of pXd-INV, as well as an increase in the H/T ratio, was observed. To disclose the binding mode of each compound and elucidate the molecular features determining its acceptor or inhibitor behaviour, ternary complexes of the inactive mutant pXd-INV-D80A with fructose and the different polyphenols were analyzed by X-ray crystallography. All the compounds bind by stacking against Trp105 and locate one of their phenolic hydroxyls making a polar linkage to the fructose O2 at 3.6-3.8 Å from the C2, which could enable the ulterior nucleophilic attack leading to transfructosylation. Binding of hydroquinone was further investigated by soaking in absence of fructose, showing a flexible site that likely allows productive motion of the intermediates. Therefore, the acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein, this flexibility being essential for the transfructosylation reaction to proceed. Finally, the binding affinity of the phenolic compounds was explained based on the two sites previously reported for pXd-INV.
多酚的酶糖化是改善其理化性质和生物利用度的一种手段。另一方面，糖苷酶可以被酚类化合物抑制。在这项工作中，我们研究了各种酚类物质（对苯二酚、羟基酪醇、没食子儿茶素没食子酸酯、儿茶酚和对硝基苯酚）作为黄伞（Xanthophyllomyces dendrorhous）β-呋喃果糖苷酶（pXd-INV）的果糖受体或抑制剂的特异性。只有对苯二酚和羟基酪醇能生成糖基化产物。对于其他酚类物质，对 pXd-INV 的水解（H）和转糖苷（T）活性均表现出抑制作用，同时 H/T 比值增加。为了揭示每种化合物的结合模式并阐明决定其受体或抑制剂行为的分子特征，通过 X 射线晶体学分析了无活性突变体 pXd-INV-D80A 与果糖和不同多酚形成的三元复合物。所有化合物都通过与色氨酸 105 堆叠结合，并使一个酚羟基与果糖 O2 形成极性键，距离 C2 为 3.6-3.8 Å，这可能使后续的亲核攻击导致转果糖基化。在没有果糖的情况下进行对苯二酚的浸泡实验，进一步研究了其结合情况，显示出一个灵活的结合位点，可能允许中间体进行产生活性的运动。因此，不同多酚的受体能力似乎与其与蛋白质形成灵活的极性键的能力有关，这种灵活性对于转果糖基化反应的进行至关重要。最后，根据先前报道的 pXd-INV 的两个结合位点，解释了酚类化合物的结合亲和力。
