Functional Characterization and Substrate Promiscuity of UGT71 Glycosyltransferases from Strawberry (Fragaria × ananassa).
Functional Characterization and Substrate Promiscuity of UGT71 Glycosyltransferases from Strawberry (Fragaria × ananassa).
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DOI：
10.1093/pcp/pcv151
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发表时间：
2015-12
影响因子：
4.9
通讯作者：
Chuankui Song;Le Gu;Jingyi Liu;Shuai Zhao;X. Hong;Katja Schulenburg;W. Schwab
中科院分区：
文献类型：
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作者：
Chuankui Song;Le Gu;Jingyi Liu;Shuai Zhao;X. Hong;Katja Schulenburg;W. Schwab
糖基化反应决定了植物天然产物的复杂性和多样性。为了表征草莓果实成熟相关的UDP依赖性糖基转移酶（UGT）的功能，我们挖掘了公开可用的野草莓基因组序列，发现了199个推定的UGT基因。从F.×ananassa中克隆了在果实成熟过程中表达水平强烈上调的候选UGT，其中6个在大肠杆菌中成功表达并进行了生化表征。UGT 75 T1显示出非常严格的底物特异性，并且在33个化合物中仅糖基化高良姜素。其他重组酶表现出广泛的底物耐受性，接受许多类黄酮，羟基香豆素，萘酚和植物激素，（+）-S-脱落酸（阿坝）。UGT 71 W2对1-萘酚表现出最高的活性，而UGT 71 A33、UGT 71 A34 a/B和UGT 71 A35优选3-羟基香豆素并形成3-和7-O-糖苷以及来自黄酮醇的二糖苷。筛选草莓生理糖苷配基文库鉴定了山萘酚、槲皮素、阿坝和三种未知的天然化合物作为UGT 71 A33、UGT 71 A34 a和UGT 71 W2的推定植物底物。RNA干扰（RNAi）介导的沉默的水果的代谢物分析表明，UGT 71 W2有助于黄酮醇，外源性物质的糖基化，并在较小程度上，阿坝，在植物。该研究表明，专家和通用UGT在草莓果实成熟过程中表达，后者可能不仅限于一种功能。
Glycosylation determines the complexity and diversity of plant natural products. To characterize fruit ripening-related UDP-dependent glycosyltransferases (UGTs) functionally in strawberry, we mined the publicly available Fragaria vesca genome sequence and found 199 putative UGT genes. Candidate UGTs whose expression levels were strongly up-regulated during fruit ripening were cloned from F.×ananassa and six were successfully expressed in Escherichia coli and biochemically characterized. UGT75T1 showed very strict substrate specificity and glucosylated only galangin out of 33 compounds. The other recombinant enzymes exhibited broad substrate tolerance, accepting numerous flavonoids, hydroxycoumarins, naphthols and the plant hormone, (+)-S-abscisic acid (ABA). UGT71W2 showed the highest activity towards 1-naphthol, while UGT71A33, UGT71A34a/b and UGT71A35 preferred 3-hydroxycoumarin and formed 3- and 7-O-glucosides as well as a diglucoside from flavonols. Screening of a strawberry physiological aglycone library identified kaempferol, quercetin, ABA and three unknown natural compounds as putative in planta substrates of UGT71A33, UGT71A34a and UGT71W2. Metabolite analyses of RNA interference (RNAi)-mediated silenced fruits demonstrated that UGT71W2 contributes to the glycosylation of flavonols, xenobiotics and, to a minor extent, of ABA, in planta. The study showed that both specialist and generalist UGTs were expressed during strawberry fruit ripening and the latter were probably not restricted to only one function in plants.