Nagashima Shigeyuki, Inagaki Rieko, Kubo Akiko, Hirotani Masao, Yoshikawa Takafumi
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, 108-8641 Tokyo, Japan.
Planta. 2004 Jan;218(3):456-9. doi: 10.1007/s00425-003-1118-0. Epub 2003 Oct 2.
A cDNA encoding UDP-glucose: formononetin 7- O-glucosyltransferase, designated UGT73F1, was cloned from yeast extract-treated Glycyrrhiza echinata L. cell-suspension cultures using probes from Scutellaria baicalensis UDP-glucose: flavonoid 7- O-glucosyltransferase. The open reading frame of the UGT73F1 cDNA encodes a 441-amino-acid protein with a predicted molecular mass of 48.7 kDa. The deduced amino acid sequence showed that the protein is related to the stress-inducible glucosyltransferases. UGT73F1 mRNA was not detected in untreated G. echinata cultures but was transiently induced by treatment with yeast extract. Recombinant UGT73F1 was expressed as a histidine-tag fusion protein in Escherichia coli and purified to near homogeneity by nickel chelate chromatography. The purified recombinant enzyme was selective for isoflavonoid, formononetin and daidzein as substrates, while flavonoids and various tested non-flavonoid compounds were poor substrates.
利用黄芩UDP - 葡萄糖：类黄酮7 - O - 葡萄糖基转移酶的探针，从酵母提取物处理的刺果甘草细胞悬浮培养物中克隆出一个编码UDP - 葡萄糖：芒柄花素7 - O - 葡萄糖基转移酶的cDNA，命名为UGT73F1。UGT73F1 cDNA的开放阅读框编码一个441个氨基酸的蛋白质，预测分子量为48.7 kDa。推导的氨基酸序列表明该蛋白质与应激诱导型葡萄糖基转移酶相关。在未处理的刺果甘草培养物中未检测到UGT73F1 mRNA，但经酵母提取物处理后会短暂诱导其表达。重组UGT73F1在大肠杆菌中作为组氨酸标签融合蛋白表达，并通过镍螯合色谱法纯化至近乎同质。纯化的重组酶对异黄酮、芒柄花素和大豆苷元作为底物具有选择性，而黄酮类化合物和各种测试的非黄酮类化合物是较差的底物。
