Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis
Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis
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DOI：
10.1111/j.1365-313x.2004.02133.x
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发表时间：
2004-08-01
期刊：
影响因子：
7.2
通讯作者：
Vogt, T
中科院分区：
文献类型：
--
作者：
Hans, J;Brandt, W;Vogt, T
在利文斯通雏菊（Dorothethanthus bellidiformis）中，甜菜色素5- 0-葡糖基转移酶（UGT 73 A5）参与甜菜色素和各种黄酮醇的区域特异性葡糖基化。基于序列比对，鉴定了可能对催化作用必需的几种氨基酸候选物。选择的氨基酸的功能性表达的蛋白质，建议参与底物结合和营业额，取代通过定点诱变。两个高度保守的氨基酸，Glu 378，位于建议的UDP-葡萄糖结合位点，和His 22，位于靠近N-末端的取代，导致酶活性的完全丧失。一个3D模型，这种区域特异性betanidin和类黄酮葡萄糖基转移酶的构建和建模的活性位点。该模型基于来自东方拟无枝酸菌（Amycolatopsis orientalis）的细菌UDP-葡萄糖依赖性葡糖基转移酶的晶体结构作为模板和产生的无效突变。为了解释所观察到的结合糖的构型的反转，半经验计算支持S-N-1反应，作为对植物天然产物葡糖基转移酶讨论的通常提出的S-N-2机制的一种合理的替代。计算出的结构数据不仅解释了质子从受体betanidin的抽象，但进一步暗示，反应机制也可能涉及一个催化三联体，与丝氨酸蛋白酶家族描述的相似之处。
In livingstone daisy (Dorotheanthus bellidiformis), betanidin 5-O-glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site-directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP-glucose binding site, and His22, located close to the N-terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP-glucose-dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an S-N-1 reaction, as one plausible alternative to the generally proposed S-N-2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.