Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases.

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions.

Reaction intensification for biocatalytic production of polyphenolic natural product di-C-ß-glucosides.

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-ß-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl ß-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4-0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-ß-glucoside synthesis from expedient substrates.