Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula - PubMed (original) (raw)

Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula

Hui Shao et al. Plant Cell. 2005 Nov.

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 UDP-glucose. 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.

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Figures

Figure 1.

Figure 1.

Structures of Three Substrates of M. truncatula UGT71G1. The known or predicted glycosylation sites are labeled with numbers: quercetin (3, 5, 7, 3′, and 4′), medicagenic acid (3 or 28), and hederagenin (3 or 28).

Figure 2.

Figure 2.

Ribbon Diagram of the Structure of UGT71G1 with Bound UDP. The N- and C-terminal domains are shown in orange and green, with the secondary structures and the N and C termini labeled. The α helices and β strands in the N- and C-terminal domains are numbered separately. The UDP molecule is shown as a ball-and-stick model colored by atom type (nitrogen, blue; carbon, yellow; oxygen, red; phosphorus, green). Figures 2, 3B, 4, 5, and 6 were prepared with MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997).

Figure 3.

Figure 3.

Comparison of M. truncatula UGT71G1 and A. orientalis GtfD. (A) Structure-based sequence alignment of M. truncatula UGT71G1 and A. orientalis GtfD. The secondary structure elements observed in the UGT71G1 structure are shown above the alignment. The UGT signature motifs are enclosed in a green box. Conserved residues are highlighted. This figure was produced with ENDscript (Gouet and Courcelle, 2002). (B) Stereo diagram showing the superimposition of the structures of UGT71G1 (molecule A; brown) and GtfD (blue; Protein Data Bank [PDB] code 1RRV).

Figure 4.

Figure 4.

Donor Molecules and Their Interactions with UGT71G1. (A) A |F obs| − |F calc| electron density omit map of bound UDP contoured at 1.5 σ is superimposed on a ball-and-stick model of the UDP molecule. (B) A |F obs| − |F calc| electron density omit map of bound UDP-glucose contoured at 1.5 σ is superimposed on a ball-and-stick model of the UDP-glucose molecule. (C) Stereo diagram showing interactions between bound UDP-glucose and UGT71G1 side chains. The structure of UDP-glucose is shown as a ball-and-stick model. Hydrogen bonding interactions are indicated by dashed lines. Interactions observed only between UDP(-galactose) and UGT71G1 in the 2.0-Å structure are indicated by green dashed lines.

Figure 5.

Figure 5.

Comparison of Donor Binding Regions of UGT71G1 and Other GT-B Fold Enzymes. Stereo diagram showing superimposition of the UGT signature motif regions of UGT71G1 (yellow), GtfD (red), MurG (green; PDB code 1F0K), OtsA (cyan; PDB code 1GZ5), and BGT (black; PDB code 1J39).

Figure 6.

Figure 6.

The Putative Acceptor Binding Pocket. Stereo diagram showing quercetin (A) and hederagenin (B) docked into the proposed binding pockets. Quercetin, hederagenin, and UDP-glucose are shown as ball-and-stick models. Some protein residues in the acceptor binding pocket are labeled and shown in cyan as bond models. Distances (Å) between the OH group of acceptors and the atom NE2 of His-22 or the atom C1′ of UDP-glucose are labeled and indicated with dashed lines.

Figure 7.

Figure 7.

Comparison of the Donor and Acceptor Regions of Functionally Characterized Plant UGTs. Sequence alignment of 39 plant UGTs, showing the acceptor binding region (first three fragments: 1 to 55, 81 to 95, and 117 to 151) and the donor binding region (last two fragments: 282 to 286 and 339 to 382). Identical residues are highlighted, and similar residues are enclosed in boxes. Residues His-22 and Asp-121 are marked with asterisks. The alignment was performed using ClustalX (Thompson et al., 1997). The plant UGTs include Medicago truncatula UGT71G1 and UGT73K1; Allium cepa UGT73G1 and UGT73J1; Aralia cordata GaT; Bellis perennis UGAT; Brassica napus SGT1; Catharanthus roseus UGT2; Citrus maxima 1,2RhaT; Crocus sativus UGTCs2; Dorotheanthus bellidiformis UGT73A5; Gentiana triflora 3′GT; Glycyrrhiza echinata UGT73F1; Nicotiana tabacum GT1a, GT2, and GT3; Phaseolus lunatus ZOG1; Phaseolus vulgaris ZOX1; Sorghum bicolor UGT85B1; Stevia rebaudiana UGT74G1, UGT76G1, and UGT85C2; and Zea mays cisZOC1 and cisZOG2. All others are from Arabidopsis thaliana.

Figure 8.

Figure 8.

Glucosylation of Quercetin by Recombinant UGT71G1. (A) HPLC trace showing the substrate quercetin (Q) and the five products (numbered 1 to 5). Solid line, complete reaction mixture; dashed line, standards for quercetin (Q), quercetin-3-_O_-glucoside (peak 3), and quercetin-4′-_O_-glucoside (peak 4). mAU, milli-absorption unit. (B) UV absorption spectra of quercetin (Q) and products 1 to 5.

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