Crown-ether-mediated crystal structures of the glycosyltransferasePaGT3 fromPhytolacca americana

Abstract

Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structurefunction relationship of PaGT3, its crystal structure was determined. The sugardonor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptorbinding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein.The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.

Structure–function relationship of terpenoid glycosyltransferases from plants

“…Currently, 24 crystal structures of plant UGTs family 1 are available these include AtUGT72B1, 53 AtUGT74F2, 54 and AtUGT89C1 55 from Arabidopsis thaliana , CtUGT78K6 from Clitoria ternatea , 56 FeUGT708C1 from Fagopyrum esculentum , 57 GgCGT from Glycyrrhiza glabra , 58 UGT73P12 from Glycyrrhiza uralensis (; 7c2x), LpCGTa and b from Landoltia punctate , 59 MtUGT71G1, 60 MtUGT85H2, 61 and MtUGT78G1 62 from Medicago truncatula , Os79 from Oryza sativa , 63 PtigS from Persicaria tinctoria , 64,65 PaGT2 66 and 3 67 from Phytolacca americana , SbCGTa and b from Scutellaria baicalensis , 59 mogrol glucosyltransferase SgUGT74AC1 and AC2 from Siraitia grosvenorii , 39,68 SrUGT76G1 from Stevia rebaudiana , 69–71 C -glycosyltransferase from Trollius chinensis , 72 VvGT1 from Vitis vinifera , 32 and ZmCGTa from Zea mays 59 (). The 3-dimensional structures show remarkable conservation in their secondary and tertiary structures.…”

Functional Characterization and Protein Engineering of a Triterpene 3‐/6‐/2′‐O‐Glycosyltransferase Reveal a Conserved Residue Critical for the Regiospecificity

“…The homology structure of AmGT8 was constructed by SWISS‐MODEL (Figure 3A, Figure S9) [33] . The crystal structure of PaGT3 (PDB ID: 6lzy) was used as the template (Figure S10) [34] . It shares a 44 % amino acid sequence identity with AmGT8.…”

Unraveling the Glucosylation of Astringency Compounds of Horse Chestnut via Integrative Sensory Evaluation, Flavonoid Metabolism, Differential Transcriptome, and Phylogenetic Analysis

“…The elucidation of several plant UGT crystals is the basis for exploring the reaction mechanism of these proteins and secondary metabolites in plants (Li et al, 2007;Yang et al, 2018;Maharjan et al, 2020). The simulation results of the three AcUGTs were all >35% and two were 50%, indicating very high feasibility.…”

“…Homology models of the AcUGT1, AcUGT22 and AcUGT26 were constructed using the three-dimensional structure of UGT74AC2 (PDB ID: 7BV3; https://www.rcsb.org/, Siraitia grosvenorii), UGT85H2 (2PQ6, Medicago truncatula) and PaGT3 (6LZX, Phytolacca Americana) as a template respectively, by the SWISS-MODEL server (http://swissmodel.expasy.org) (Li et al, 2007(Li et al, , 2021Maharjan et al, 2020). UDP-glucose or UDP-rhamnose and quercetin were docked with the model structure of AcUGTs using the Igemdock 2.1 docking program (http://gemdock.life.nctu.edu.tw/dock/igemdock.php).…”