Flavonoid natural products are well known for their beneficial antimicrobial, antitumor, and anti-inflammatory properties, however, some of these natural products often are rhamnosylated, which severely limits their bioavailability. The lack of endogenous rhamnosidases in the human GI tract not only prevents many of these glycosylated compounds from being of value in functional foods but also limits the modification of natural product libraries being tested for drug discovery. RHA-P is a catalytically efficient, thermostable α-l-rhamnosidase from the marine bacterium Novosphingobium sp. PP1Y that selectively hydrolyzes α-1,6 and α-1,2 glycosidic linkages between a terminal rhamnose and a flavonoid moiety. This work reports the 2.2 Å resolution crystal structure of RHA-P, which is an essential step forward in the characterization of RHA-P as a potential catalyst to increase the bioavailability of rhamnosylated natural compounds. The structure shows highly conserved rhamnose- and calcium-binding residues in a shallow active site that is housed in the (β/α)8 domain. In comparison to BT0986 (pdbID: 5MQN), the only known structure of an RHA-P homolog, the morphology, electrostatic potentials and amino acid composition of the substrate binding pocket are significantly different, offering insight into the substrate preference of RHA-P for glycosylated aryl compounds such as hesperidin, naringin, rutin, and quercitrin, over polysaccharides, which are preferred by BT0986. These preferences were further explored by using in silico docking, the results of which are consistent with the known kinetic data for RHA-P acting on different rhamnosylated flavonoids. Due to its promiscuity, relative thermostability compared to other known rhamnosidases, and catalytic efficiency even in significant concentrations of organic solvents, RHA-P continues to show potential for biocatalytic applications.

中文翻译：

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Novosphingobium sp。的α-L鼠李糖苷酶RHA-P的晶体结构和对底物特异性的了解。PP1Y。

类黄酮天然产物以其有益的抗微生物，抗肿瘤和抗炎特性而闻名，但是，其中一些天然产物经常被鼠李糖基化，这严重限制了它们的生物利用度。人的胃肠道中缺乏内源鼠李糖苷酶，不仅阻止了许多这些糖基化化合物在功能性食品中的价值，而且还限制了用于药物发现测试的天然产物库的修饰。RHA-P是来自海洋细菌Novosphingobium sp。的催化有效的，热稳定的α-1-鼠李糖苷酶。选择性水解末端鼠李糖和类黄酮部分之间的α-1,6和α-1,2糖苷键的PP1Y。这项工作报告了RHA-P的2.2Å分辨率晶体结构，这是表征RHA-P作为增加鼠李糖基化天然化合物生物利用度的潜在催化剂的重要一步。该结构在（β/α）8结构域中的浅活性位点显示了高度保守的鼠李糖和钙结合残基。与BT0986（pdbID：5MQN）相比，RHA-P同源物的唯一已知结构，底物结合口袋的形态，静电势和氨基酸组成明显不同，从而可以更深入地了解RHA-P对底物的偏好糖基化的芳基化合物（如橙皮苷，柚皮苷，芦丁和槲皮素）优于多糖，而BT0986则更优选多糖。通过计算机对接进一步探索了这些首选项，结果与已知的RHA-P作用于不同鼠李糖基黄酮类化合物的动力学数据一致。由于其混杂性，与其他已知鼠李糖苷酶相比的相对热稳定性以及即使在高浓度的有机溶剂中也具有催化效率，RHA-P继续显示出生物催化应用的潜力。