Uridine diphosphate-dependent glycosyltransferases (UGTs) catalyze the transfer of a diversity of sugars to several acceptor molecules and often exhibit distinct substrate specificity. Modulation of glycosyltransferases for increased catalytic activity and altered substrate or product specificity are the key manipulations for the biotechnological use of glycosyltransferases in various biosynthetic processes. Here, we have engineered the binding pocket of three previously characterized Vitis vinifera glycosyltransferases, UGT88F12, UGT72B27 and UGT92G6, by structure-guided in silico mutagenesis to facilitate the interactions of active site residues with flavonol glucosides and thus modify substrate specificity and activity. Site-directed mutagenesis at selected sites, followed with liquid chromatography–mass spectrometry based activity assays, exhibited that mutant UGTs were altered in product selectivity and activity as compared to the wild-type enzymes. Mutant UGTs produced larger amounts of flavonol di-monosaccharide glucosides, which imply that the mutations led to structural changes that increased the volume of the binding pocket to accommodate a larger substrate and to release larger products at ease. Mutants showed increased activity and modified product specificity. Thus, structure-based systematic mutations of the amino acid residues in the binding pocket can be explored for the generation of engineered UGTs for diverse biotechnological applications.

中文翻译：

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葡萄葡糖基转移酶的半定量设计和工程设计，可增强活性和改善产品的选择性

尿苷二磷酸依赖性糖基转移酶（UGTs）催化多种糖类向几个受体分子的转移，并且通常表现出不同的底物特异性。为了增加催化活性和改变底物或产物特异性而对糖基转移酶进行调节是糖基转移酶在各种生物合成过程中的生物技术用途的关键操作。在这里，我们设计了三个先前表征的葡萄（Vitis vinifera）的结合袋糖基转移酶UGT88F12，UGT72B27和UGT92G6，通过结构指导的计算机诱变，促进活性位点残基与黄酮醇苷的相互作用，从而修饰底物特异性和活性。在选定的位点进行定点诱变，然后进行基于液相色谱-质谱的活性测定，结果表明，与野生型酶相比，突变型UGT在产物选择性和活性上均发生了变化。突变的UGT产生了大量的黄酮醇二单糖苷，这表明该突变导致结构变化，增加了结合袋的体积，可以容纳更大的底物并轻松释放更大的产物。突变体显示出增加的活性和修饰的产物特异性。因此，