Wasabi (Eutrema japonicum) is a perennial plant native to Japan that is used as a spice because it contains isothiocyanates. It also contains an isosaponarin, 4'-O-glucosyl-6-C-glucosyl apigenin, in its leaves, which has received increasing attention in recent years for its bioactivity, such as its promotion of type-I collagen production. However, its biosynthetic enzymes have not been clarified. In this study, we partially purified a C-glucosyltransferase (CGT) involved in isosaponarin biosynthesis from wasabi leaves and identified the gene coding for it (WjGT1). The encoded protein was similar to UGT84 enzymes and was named UGT84A57. The recombinant enzyme of WjGT1 expressed in Escherichia coli showed C-glucosylation activity toward the 6-position of flavones such as apigenin and luteolin. The enzyme also showed significant activity toward flavonols, but trace or no activity toward flavone 4'-O-glucosides, suggesting that isosaponarin biosynthesis in wasabi plants would proceed by 6-C-glucosylation of apigenin, followed by its 4'-O-glucosylation. Interestingly, the enzyme showed no activity against sinapic acid or p-coumaric acid, which are usually the main substrates of UGT84 enzymes. The accumulation of WjGT1 transcripts was observed mainly in the leaves and flowers of wasabi, in which C-glucosylflavones were accumulated. Molecular phylogenetic analysis suggested that WjGT1 acquired C-glycosylation activity independently from other reported CGTs after the differentiation of the family Brassicaceae.

中文翻译：

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芥末（Eutrema japonicum）中异叶皂苷的生物合成中涉及的芹菜素6-C-葡萄糖基转移酶的鉴定与表征。

山葵（Eutrema japonicum）是日本本土的多年生植物，由于含有异硫氰酸盐而被用作香料。它的叶子中还含有异叶皂甙元，即4'-O-葡萄糖基-6-C-葡萄糖基芹菜素，近年来由于其生物活性（例如促进I型胶原蛋白的产生）而受到越来越多的关注。然而，其生物合成酶尚未阐明。在这项研究中，我们从芥末叶中部分纯化了参与异叶皂苷生物合成的C-葡萄糖基转移酶（CGT），并鉴定了编码它的基因（WjGT1）。编码的蛋白质类似于UGT84酶，被命名为UGT84A57。在大肠杆菌中表达的WjGT1重组酶显示出对黄酮（如芹菜素和木犀草素）的6位具有C-糖基化活性。该酶还显示出对黄酮醇的显着活性，但对黄酮4'-O-葡糖苷没有或没有活性，表明芥末植物中异皂苷的生物合成将通过芹菜素的6-C-葡糖基化，然后是其4'-O-葡糖基化来进行。 。有趣的是，该酶对通常为UGT84酶主要底物的芥子酸或对香豆酸没有活性。主要在芥末的叶子和花朵中观察到WjGT1转录物的积累，其中积累了C-葡萄糖基黄酮。分子系统发育分析表明，在十字花科的分化之后，WjGT1独立于其他报道的CGT获得了C-糖基化活性。提示芥末植物中异叶皂苷的生物合成将通过芹菜素的6-C-糖基化，然后是其4'-O-糖基化来进行。有趣的是，该酶对通常为UGT84酶主要底物的芥子酸或对香豆酸没有活性。主要在芥末的叶子和花朵中观察到WjGT1转录物的积累，其中积累了C-葡萄糖基黄酮。分子系统发育分析表明，在十字花科的分化之后，WjGT1独立于其他报道的CGT获得了C-糖基化活性。提示芥末植物中异叶皂苷的生物合成将通过芹菜素的6-C-糖基化，然后是其4'-O-糖基化来进行。有趣的是，该酶对通常为UGT84酶主要底物的芥子酸或对香豆酸没有活性。主要在芥末的叶子和花朵中观察到WjGT1转录物的积累，其中积累了C-葡萄糖基黄酮。分子系统发育分析表明，在十字花科的分化之后，WjGT1独立于其他报道的CGT获得了C-糖基化活性。主要在芥末的叶子和花朵中观察到WjGT1转录物的积累，其中积累了C-葡萄糖基黄酮。分子系统发育分析表明，在十字花科的分化之后，WjGT1独立于其他报道的CGT获得了C-糖基化活性。主要在芥末的叶子和花朵中观察到WjGT1转录物的积累，其中积累了C-葡萄糖基黄酮。分子系统发育分析表明，在十字花科的分化之后，WjGT1独立于其他报道的CGT获得了C-糖基化活性。