Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris
Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris
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DOI：
10.1016/j.phytochem.2006.06.026
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发表时间：
2006-08-01
期刊：
影响因子：
3.8
通讯作者：
Vogt, Thomas
中科院分区：
文献类型：
--
作者：
Isayenkova, Judith;Wray, Victor;Vogt, Thomas
从苋菜（Beta vulgaris, Amaranthaceac）细胞悬浮培养的cDNA文库中分离到两个编码黄酮类特异性糖基转移酶的全长cDNA，分别为UGT73A4和UGT71F1。它们对桃面花（Dorotheanthus bellidiformis）中的位置特异性betanidin和类黄酮糖基转移酶以及来自不同植物科的具有相似底物特异性的酶具有高度的一致性。序列的开放阅读框编码476个（UGT73A4）和492个（UGT71F1）氨基酸的蛋白，计算得到的分子质量分别为54.07 kDa和54.39 kDa，等电点分别为5.8和5.6。这两种酶在大肠杆菌中分别以His-和gst标记蛋白的形式功能性表达。它们表现出广泛的底物特异性，但具有明显的区域选择性，可以糖基化各种黄酮醇、黄酮、黄烷酮和香豆素。UGT73A4在黄酮类化合物中表现出对4'-和7-OH位置的偏好，而UGT71F1则优先对3-或7-OH位置进行糖基化。两种酶对甜菜素的糖基化作用也均较低。本文讨论了从白杨幼苗叶片中分离的几种o糖基化牡荆素衍生物和从白杨组织培养中获得的芦丁作为UGT73A4和UGT71F1的潜在内源产物。对植物天然产物糖基转移酶的进化和特异性进行了分析。(c) 2006 Elsevier Ltd.版权所有。
Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceac) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07 kDa and 54.39 kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4'- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases., (c) 2006 Elsevier Ltd. All rights reserved.