添加O-连接的N-乙酰葡糖胺(O-GlcNAc)是活性分子的重要修饰,如蛋白质,碳水化合物,和天然产品。然而,由于缺乏糖基转移酶,用GlcNAc修饰的萜类糖苷衍生物的合成仍然是一项具有挑战性的任务,催化GlcNAc向萜类化合物转移的关键酶。在这项研究中,我们证明了酶突变体UGT74AC1T79Y/L48M/R28H/L109I/S15A/M76L/H47R将GlcNAc从尿苷二磷酸(UDP)-GlcNAc有效地转移到多种萜类化合物中。这种强大的酶被用来合成萜类化合物的GlcNAc修饰衍生物,包括莫格罗,甜菊醇,穿心莲内酯,原人参二醇,甘草次酸,熊果酸,和桦木酸的第一次。为了解开UDP-GlcNAc识别的机制,我们确定了与UDP-GlcNAc复合的灭活突变体UGT74AC1His18A/Asp111A的X射线晶体结构,分辨率为1.66。通过分子动力学模拟和活性分析,我们揭示了UDP-GlcNAc识别的分子机制和催化重要的氨基酸。总的来说,这项研究不仅提供了一种能够使天然产物糖多样化的有效生物催化剂,而且还阐明了糖基转移酶识别UDP-GlcNAc的结构基础。
The addition of the O-linked N-acetylglucosamine (O-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products. However, the synthesis of terpenoid glycoside derivatives decorated with GlcNAc remains a challenging task due to the absence of glycosyltransferases, key enzymes for catalyzing the transfer of GlcNAc to terpenoids. In this study, we demonstrated that the enzyme mutant UGT74AC1T79Y/L48M/R28H/L109I/S15A/M76L/H47R efficiently transferred GlcNAc from uridine diphosphate (UDP)-GlcNAc to a variety of terpenoids. This powerful enzyme was employed to synthesize GlcNAc-decorated derivatives of terpenoids, including mogrol, steviol, andrographolide, protopanaxadiol, glycyrrhetinic acid, ursolic acid, and betulinic acid for the first time. To unravel the mechanism of UDP-GlcNAc recognition, we determined the X-ray crystal structure of the inactivated mutant UGT74AC1His18A/Asp111A in complex with UDP-GlcNAc at a resolution of 1.66 Å. Through molecular dynamic simulation and activity analysis, we revealed the molecular mechanism and catalytically important amino acids directly involved in the recognition of UDP-GlcNAc. Overall, this study not only provided a potent biocatalyst capable of glycodiversifying natural products but also elucidated the structural basis for UDP-GlcNAc recognition by glycosyltransferases.