Structural insights into the catalytic mechanism of the phenylethanoid glycoside rhamnosyltransferase UGT79G15 from Rehmannia glutinosa

Abstract

Phenylethanoid glycosides (PhGs) are a group of important natural products widely distributed in medicinal plants and known for their remarkable pharmacological properties. Uridine diphosphate (UDP) glycosyltransferase 79G15 (UGT79G15) from Rehmannia glutinosa catalyzes the conversion of osmanthuside A to osmanthuside B, a key intermediate in the PhG biosynthetic pathway, through the formation of a (1→3) glycosidic bond. In this study, we present the crystal structures of UGT79G15 in its apo form, UDP-bound form, and, notably, its ternary complex containing UDP and a mimic acceptor, forsythiaside A, within its active site. Structural and comparative analyses revealed that UGT79G15 possesses a distinctive funnel-shaped acceptor-binding pocket with a small auxiliary cavity capable of accommodating the 4'-hydroxycinnamoyl group of PhGs, explaining the enzyme's regiospecificity toward the 3'-OH of the acceptor. Additional structural examination and site-directed mutagenesis identified key residues that recognize and stabilize UDP-rhamnose and the sugar acceptor. Among the variants generated, I204W exhibits enhanced catalytic efficiency for osmanthuside A conversion, reaching up to 2.2-fold higher activity than the wild type. This study provides mechanistic insight into the donor specificity and acceptor regioselectivity of PhG 1,3-rhamnosyltransferase and expands the structural understanding of plant UGTs.

Keywords: acceptor regiospecificity; crystal complex; funnel-shaped pocket; phenylethanoid glycosides; rhamnosyltransferase.

Figure 1

Function and crystal structure characteristics of UGT79G15. (A) Schematic representation of the reaction catalyzed by UGT79G15. (B) Ribbon representation of the UGT79G15 crystal structure showing forsythiaside A and UDP at the active site with a narrow entrance. The N-terminal domain is shown in green, and the C-terminal domain in blue. (C) Superposition of the structures of UGT79G15_apo (orange), UGT79G15_UDP (green), and UGT79G15_UDP_FA (lilac). (D) Acceptor-binding pocket of UGT79G15.

Figure 2

Analysis of the catalytic mechanism of UGT79G15. (A) Docking structure of UGT79G15_UDP-Rha_forsythiaside A. (B) Docking structure of UGT79G15_UDP-Rha_osmanthuside A. (C) Residues interacting with UDP-Rha and forsythiaside A. (D) Residues interacting with UDP-Rha and osmanthuside A. (E) Proposed catalytic mechanism of UGT79G15. (F) Validation of the catalytic site of UGT79G15 using osmanthuside A or forsythiaside A as substrates and UDP-Rha as the sugar donor. N.D., not detected.

Figure 3

UDP molecule and its interaction with UGT79G15. (A) Comparison of the UDP-binding pockets of UGT79G15_apo (wheat) and UGT79G15_UDP (light cyan). (B) Comparison of the distances between UDP and surrounding residues in UGT79G15_apo (wheat) and UGT79G15_UDP (light cyan). (C) Two-dimensional interaction analysis of UDP and its surrounding residues.

Figure 4

Substrate-binding pocket of UGT79G15. (A) Binding mode of UDP and forsythiaside A within the substrate-binding channel of UGT79G15. (B) Residues interacting with UDP and forsythiaside A around the binding pocket. (C) Two-dimensional interaction analysis of forsythiaside A and its surrounding residues.

Figure 5

Cross-sectional diagrams of acceptor-binding pockets in UGT79G15, UGT89C1, and UGT74AN3. (A) Funnel-shaped pocket of UGT79G15 with the substrate forsythiaside A bound inside. (B) V-shaped pocket of UGT89C1 with the substrate quercetin bound inside. (C) U-shaped pocket of UGT74AN3 with the substrate resibufogenin bound inside.

Figure 6

Catalytic activities of UGT79G15 and its mutants. (A) Conversion rate of the glycosylated product obtained using osmanthuside A as the substrate and UDP-Rha as the sugar donor. (B) Conversion rate of the glycosylated product obtained using forsythiaside A as the substrate and UDP-Rha as the sugar donor.