Crystal Structures of a Multifunctional Triterpene/Flavonoid Glycosyltransferase from Medicago truncatula

Abstract

Glycosylation is a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of glycosyltransferases (GTs) in most plant species studied to date. We present crystal structures of the UDP flavonoid/triterpene GT UGT71G1 from Medicago truncatula bound to UDP or UDPglucose. The structures reveal the key residues involved in the recognition of donor substrate and, by comparison with other GT structures, suggest His-22 as the catalytic base and Asp-121 as a key residue that may assist deprotonation of the acceptor by forming an electron transfer chain with the catalytic base. Mutagenesis confirmed the roles of these key residues in donor substrate binding and enzyme activity. Our results provide an initial structural basis for understanding the complex substrate specificity and regiospecificity underlying the glycosylation of plant natural products and other small molecules. This information will direct future attempts to engineer bioactive compounds in crop plants to improve plant, animal, and human health and to facilitate the rational design of GTs to improve the storage and stability of novel engineered bioactive compounds.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

“…The OleD Ser132 equivalent in the plant flavonoid GT VvGT1 (Thr141) forms a hydrogen bond with the C-6 hydroxyl group of UDP-2-deoxy-2-fluoroglucose (33,34). Lack of a hydrogen bond donor/acceptor at this position may explain the large increases in activity observed in previous studies (17) and in this investigation, particularly with C-6 modified sugars.…”

“…In the OleD crystal structure, Ala242 follows Ser241, which forms a hydrogen bond to the α-phosphate of the UDP acceptor in the context of a GT-catalyzed reverse reaction (this interaction was also observed in the VvGT1 structure) (14,27,33). Thus, hydrophobic mutations at the adjacent residue may influence NDP acceptor and/or sugar moiety interactions after transfer from a 2-chloro-4-nitrophenyl glycoside donor.…”

A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

“…Investigation of the 3D-structures of betanidin 5-O-glucosyltransferase (B5GT) from Dorotheanthus bellidiformis [17] and cyanohydrin glucosyltransferase from Sorghum bicolor [18] by homology modeling, and of isoflavonoid 3 0 -O-glucosyltransferase from Medicago truncatula [19,20] and flavonoid 3-O-glucosyltransferase from Vitis vinifera [21] by X-ray crystallography, revealed the role of specific conserved amino acid residues in the PSPG-box that constitute the donor-sugar binding pockets. However, the roles of less well conserved amino acids within the motif that may determine the characteristics unique to particular enzymes such as substrate recognition and catalytic potential have been less closely examined.…”

Characterization and engineering of the bifunctional N - and O -glucosyltransferase involved in xenobiotic metabolism in plants

“…Although the SGTs in groups D and H had comparable OGT activities, those in group L showed a preference for the thiol acceptor. Of these enzymes, only UGT74B1 was known to be an SGT because of its glucosylating activity toward thiohydroximate precursors of glucosinolates (10). The other five UGTs had been shown to have OGT activity toward natural products, with UGT74F1 and UGT84B1 conjugating anthranilic acid and indole acetic acid, respectively.…”

“…3 B and C). In the majority of inverting UGTs (10,12,18), the equivalent histidine forms an Oacceptor-His-Asp triad (Fig. 3C), analogous to the geometry of the Ser-His-Asp triad of serine hydrolases (see ref.…”