Institute of Bioinformatics and Biotechnology (IBB), Savitribai Phule Pune University (Formerly University of Pune), Ganeshkhind Road, Pune 411007, Maharashtra, India.
Biotechnology of Natural Products, Technische Universität München, 85354 Freising, Liesel-Beckmann-Str. 1, Germany.
Glycobiology. 2019 Oct 21;29(11):765-775. doi: 10.1093/glycob/cwz056.
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.
尿苷二磷酸依赖性糖基转移酶（UGTs）催化多种糖向几个受体分子的转移，通常表现出不同的底物特异性。糖基转移酶的调节以提高催化活性和改变底物或产物特异性是在各种生物合成过程中生物技术应用糖基转移酶的关键操作。在这里，我们通过基于结构的计算诱变工程化了三个先前表征的葡萄苷基转移酶，UGT88F12、UGT72B27 和 UGT92G6 的结合口袋，以促进活性位点残基与类黄酮葡萄糖苷的相互作用，从而改变底物特异性和活性。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，突变体 UGT 在产物选择性和活性方面发生了改变。与野生型相比，突变体 UGT 产生了更多的类黄酮二单糖苷，这意味着突变导致结构发生变化，增加了结合口袋的体积，以容纳更大的底物，并更容易释放更大的产物。突变体显示出增加的活性和改变的产物特异性。因此，可以探索基于结构的结合口袋中氨基酸残基的系统突变，以产生用于各种生物技术应用的工程化 UGT。
