Crown-ether-mediated crystal structures of the glycosyltransferase PaGT3 from Phytolacca americana
Crown-ether-mediated crystal structures of the glycosyltransferase PaGT3 from Phytolacca americana
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DOI：
10.1107/s2059798320005306
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发表时间：
2020-06-01
影响因子：
2.2
通讯作者：
Inoue, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Maharjan, Rakesh;Fukuda, Yohta;Inoue, Tsuyoshi
尿苷二磷酸糖基转移酶（UGT）是参与小分子糖基化的普遍存在的酶。由于糖基化改善了疏水性化合物的水溶性和稳定性，因此对使用UGT合成难溶性化合物的糖苷的兴趣正在增加。虽然在UGT中糖供体识别是保守的，存在植物次级产物糖基转移酶（PSPG）基序，但由于受体结合区周围的低序列同一性，糖受体识别的基础和产物的区域选择性知之甚少。PaGT 3是一种来自美洲商陆的糖基转移酶，可以使一系列受体糖基化。为了说明PaGT 3的结构-功能关系，测定了其晶体结构。通过与其他UGT的结构比较，确认了PaGT 3中的糖供体和糖受体结合口袋。PaGT 3的关键特征是在疏水受体结合口袋周围存在较长的环区域，这导致了柔性和较宽的受体结合口袋。在这项研究中，通过与18-冠-6醚或15-冠-5醚共结晶来生长PaGT 3晶体。观察到冠醚分子中的不对称单元与金属离子形成络合物，该络合物在两侧由来自两个分子的蛋白质的Glu 238的主链O原子配位。冠醚-金属复合物类似于分子胶，将两个PaGT 3分子粘在一起以促进晶体生长。因此，这一结果提供了一个深入了解的底物识别策略在PaGT 3的糖基转移酶的研究。此外，它表明，冠醚-金属离子配合物可以用作分子胶的蛋白质结晶。
Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structure-function relationship of PaGT3, its crystal structure was determined. The sugar-donor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptor-binding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein. The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.