Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants
Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants
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DOI：
10.1073/pnas.0706421104
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发表时间：
2007-12-18
影响因子：
11.1
通讯作者：
Edwards, Robert
中科院分区：
文献类型：
--
作者：
Brazier-Hicks, Melissa;Offen, Wendy A.;Edwards, Robert
植物体内污染物和农药代谢物的糖基化作用控制着它们的生物活性和后续化学残留物的形成。模式植物拟南芥含有100个糖基转移酶(GT；100)，专用于小分子结合，其中44个酶催化氯代酚的O-糖基化，只有一个酶UGT72B1对氯代苯胺具有明显的N-糖基化活性。UGT72B1是一种具有双功能的O-葡萄糖转移酶(OGT)和N-葡萄糖转移酶(NGT)。为了研究这种独特的双重活性，蛋白质的结构被解析，分辨率高达1.45埃，包括具有完整供体类似物和三氯苯酚受体的Michaelis络合物。用一种仅具有OGT活性的甘蓝型油菜同源酶(BnUGT)进行诱变和结构域洗牌，探讨了O/N专一性的催化机理和基础。仅在D312N和F315Y两个位置的BnUGT突变就具有高水平的NGT活性。分子模拟揭示了这些残基与UGT72B1上的H19的连接性，其突变专门定义了拟南芥酶中的NGT活性。这些结果揭示了植物GT与非自然底物的结合，突出了这类酶的催化可塑性，以及设计不寻常和理想的转移到氮基受体的能力。
The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable N-glucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 angstrom, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.