Regioselective Glycosylation of Polyphenols by Family 1 Glycosyltransferases: Experiments and Simulations.

Family 1 glycosyltransferases (GT1s, UGTs) form natural product glycosides with exquisite control over regio- and stereoselectivity, representing attractive biotechnological targets. However, regioselectivity cannot be predicted and large-scale activity assessment efforts of UGTs are commonly performed via mass spectrometry or indirect assays that are blind to regioselectivity. Here, we present a large high performance liquid chromatography screening discriminating between regioisomeric products of 40 diverse UGTs (28.6% average pairwise sequence identity) against 32 polyphenols, identifying enzymes able to reach high glycosylation yields (≥90% in 24 h) in 26/32 cases. In reactions with >50% yield, we observed perfect regioselectivity for 47% (75/158) on polyphenols presenting two hydroxyl groups and for 30% (43/143) on polyphenols presenting ≥3 hydroxyl groups. Moreover, we developed a nuclear magnetic resonance-based procedure to identify the site of glycosylation directly on enzymatic mixtures. We further selected seven regiospecific reactions catalyzed by four enzymes on five dihydroxycoumarins. We characterized the four enzymes, showing that temperature optima are functions of the acceptor substrate, varying by up to 20 °C for the same enzyme. Furthermore, we performed short molecular dynamics simulations of 311 ternary complexes (UGT, UDP-Glc, and glycosyl acceptor) to investigate the molecular basis for regioselectivity. Interestingly, it appeared that most UGTs can accommodate acceptors in configurations favorable to the glycosylation of either hydroxyl. In contrast, evaluation of hydroxyl nucleophilicity appeared to be a strong predictor of the hydroxyl predominantly glycosylated by most enzymes.

Promiscuous Yet Specific: A Methionine-Aromatic Interaction Drives the Reaction Scope of the Family 1 Glycosyltransferase GmUGT88E3 from Soybean.

Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified GmUGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.e., promiscuous toward acceptors while regiospecific. Using high-field NMR, kinetic characterization, molecular dynamics simulations, and mutagenesis studies, we uncovered that the main molecular determinant of GmUGT88E3 specificity is a methionine-aromatic bridge, an interaction often present in protein structures but never reported for enzyme-substrate interactions. Here, mutating Met127 led to inactive proteins or 100-fold reduced activity.

Sequence mining yields 18 phloretin C-glycosyltransferases from plants for the efficient biocatalytic synthesis of nothofagin and phloretin-di-C-glycoside.

C-glycosyltransferases (C-GTs) offer selective and efficient synthesis of natural product C-glycosides under mild reaction conditions. In contrast, the chemical synthesis of these C-glycosides is challenging and environmentally harmful. The rare occurrence of C-glycosylated compounds in Nature, despite their stability, suggests that their biosynthetic enzymes, C-GTs, might be scarce. Indeed, the number of characterized C-GTs is remarkably lower than O-GTs. Therefore, discovery efforts are crucial for expanding our knowledge of these enzymes and their efficient application in biocatalytic processes. This study aimed to identify new C-GTs based on their primary sequence. 18 new C-GTs were discovered, 10 of which yielded full conversion of phloretin to its glucosides. Phloretin is a dihydrochalcone natural product, with its mono-C-glucoside, nothofagin, having various health-promoting effects. Several of these enzymes enabled highly selective production of either nothofagin (UGT708A60 and UGT708F2) or phloretin-di-C-glycoside (UGT708D9 and UGT708B8). Molecular docking simulations, based on structural models of selected enzymes, showed productive binding modes for the best phloretin C-GTs, UGT708F2 and UGT708A60. Moreover, we characterized UGT708A60 as a highly efficient phloretin mono-C glycosyltransferase (kcat  = 2.97 s-1 , KM  = 0.1 µM) active in non-buffered, dilute sodium hydroxide (0.1-1 mM). We further investigated UGT708A60 as an efficient biocatalyst for the bioproduction of nothofagin.