02339nas a2200217   4500008004100000022001100041245014200052210006900194260000900263300001500272490000700287520156300294100002101857700001601878700002301894700001801917700001801935700002101953700002101974856012601995       2018                        eng d  a32078100aStructural and functional analysis of UGT92G6 suggests an evolutionary link between mono- and disaccharide glycoside-forming transferases0 aStructural and functional analysis of UGT92G6 suggests an evolut  cAPR   a 862–8750 v593 a<p>Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.</p>1 aHuang, Fong-Chin1 aGiri, Ashok1 aDaniilidis, Melina1 aSun, Guangxin1 aHärtl, Katja1 aHoffmann, Thomas1 aSchwab, Wilfried  uhttps://library.ncl.res.in/content/structural-and-functional-analysis-ugt92g6-suggests-evolutionary-link-between-mono-and02450nas a2200289   4500008004100000022001400041245012900055210006900184260000800253300001200261490000700273520149600280653001301776653002401789653002301813653001601836653001601852100001801868700002001886700002301906700001801929700002701947700001601974700002101990700002102011856012802032       2019                        eng d  a0959-665800aSemirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity0 aSemirational design and engineering of grapevine glucosyltransfe  cNOV  a765-7750 v293 a<p>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.</p>
10aflavonol10aGlycosyltransferase10ain silico analysis10aMutagenesis10aselectivity1 aJoshi, Rakesh1 aTrinkl, Johanna1 aHaugeneder, Annika1 aHaertl, Katja1 aFranz-Oberdorf, Katrin1 aGiri, Ashok1 aHoffmann, Thomas1 aSchwab, Wilfried  uhttps://library.ncl.res.in/content/semirational-design-and-engineering-grapevine-glucosyltransferases-enhanced-activity-and