Functional Identification of HhUGT74AG11-A Key Glycosyltransferase Involved in Biosynthesis of Oleanane-Type Saponins in Hedera helix

Abstract

Hedera helix is a traditional medicinal plant. Its primary active ingredients are oleanane-type saponins, which have extensive pharmacological effects such as gastric mucosal protection, autophagy regulation actions, and antiviral properties. However, the glycosylation-modifying enzymes responsible for catalyzing oleanane-type saponin biosynthesis remain unidentified. Through transcriptome, cluster analysis, and PSPG structural domain, this study preliminarily screened four candidate UDP-glycosyltransferases (UGTs), including Unigene26859, Unigene31717, CL11391.Contig2, and CL144.Contig9. In in vitro enzymatic reactions, it has been observed that Unigene26859 (HhUGT74AG11) has the ability to facilitate the conversion of oleanolic acid, resulting in the production of oleanolic acid 28-O-glucopyranosyl ester. Moreover, HhUGT74AG11 exhibits extensive substrate hybridity and specific stereoselectivity and can transfer glycosyl donors to the C-28 site of various oleanane-type triterpenoids (hederagenin and calenduloside E) and the C-7 site of flavonoids (tectorigenin). Cluster analysis found that HhUGT74AG11 is clustered together with functionally identified genes AeUGT74AG6, CaUGT74AG2, and PgUGT74AE2, further verifying the possible reason for HhUGT74AG11 catalyzing substrate generalization. In this study, a novel glycosyltransferase, HhUGT74AG11, was characterized that plays a role in oleanane-type saponins biosynthesis in H. helix, providing a theoretical basis for the production of rare and valuable triterpenoid saponins.

Keywords: Hedera helix; UDP-glycosyltransferase; biosynthesis; oleanane-type saponin; substrate hybridity.

Figure 1

The inferences derived from the biosynthetic pathway of oleanane-type saponins in H. helix and the genes participating in this pathway. (a) Simplified pathway for the production of oleanane-type saponins is presented. (b) Hierarchical clustering is conducted on the expression patterns of 82 unigenes, and the standardized FPKM values are subjected to Z-Score transformation (Table S2). L1–L3 and R1–R3 represent three repetitions in leaves and roots, respectively. DXS, 1-deoxy-D-xylulose-5-phosphate-synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductase; MEP-CT, 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase; CMK, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol kinase; MECDPS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; HMBPPS, 1-hydroxy-2-methyl-2-E-butenyl-4-diphosphate synthase; HDR, 1-hydroxy-2methy-3-E-butenyl-4-diphosphate reductase; AAC, acetoacetyl-CoA thiolase; HMGS, 3-hydroxy-3-methyl glutaryl coenzyme A synthase; HMGR, 3-hydroxy-3-methyl glutaryl coenzyme A reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate 5-diphosphatcdecarboxylase; IDI, isopentenyl diphosphate isomerase; FPS, farnesyldiphosphate synthase; SS, squalene synthase; SE, squalene epoxidase; OSC, oxidosqualene cyclase; CYP450, cytochrome P450; UGT, UDP-glycosyltransferase.

Figure 2

The cluster analysis and PSPG motif alignment of candidate UGTs and Arabidopsis thaliana AtUGT74 subfamily members. (a) The construction of the phylogenetic tree of candidate UGTs and AtUGT74 subfamily members using MEGA X V1.2.6 software. (b) Motif alignment between candidate UGTs and AtUGT74 with MEME. H. helix candidate UGTs are in red font.

Figure 3

The in vitro enzymatic activity of candidate UGTs detected by UPLC-ESI-MS. (a) Extracted ion chromatograms (EICs) for 1 oleanolic acid (m/z = 454.90–455.90, RT = 4.96 min) and 1a oleanolic acid 28-O-glucopyranosyl ester (m/z = 616.90–617.90, RT = 7.03 min). (b) MS/MS fragmentation of the product peaks by HhUGT74AG11 + 1 compared with fragmentation results of the Std. 1a ([(-)-mode], m/z = 617.52).

Figure 4

The UPLC-ESI-MS analysis detected the substrate hybridity of HhUGT74AG11. (a) HhUGT74AG11 functions in the production of various oleanane-type saponins. (b) The identified compounds were as follows: 2 hederagenin (m/z = 470.90–471.90, RT = 4.69 min) and 2a hederagenin 28-O-glucopyranosyl ester (m/z = 632.90–633.90, RT = 6.11 min); (c) 3 echinocystic acid (m/z = 470.90–471.90, RT = 4.71 min) and 3a echinocystic acid 28-O-glucopyranosyl ester (m/z = 632.90–633.90, RT = 6.38 min); (d) 4 calenduloside E (m/z = 630.90–631.90, RT = 4.29 min) and 4a chikusetsusaponin IVa (m/z = 792.90–793.90, RT = 4.68 min).

Figure 4

The UPLC-ESI-MS analysis detected the substrate hybridity of HhUGT74AG11. (a) HhUGT74AG11 functions in the production of various oleanane-type saponins. (b) The identified compounds were as follows: 2 hederagenin (m/z = 470.90–471.90, RT = 4.69 min) and 2a hederagenin 28-O-glucopyranosyl ester (m/z = 632.90–633.90, RT = 6.11 min); (c) 3 echinocystic acid (m/z = 470.90–471.90, RT = 4.71 min) and 3a echinocystic acid 28-O-glucopyranosyl ester (m/z = 632.90–633.90, RT = 6.38 min); (d) 4 calenduloside E (m/z = 630.90–631.90, RT = 4.29 min) and 4a chikusetsusaponin IVa (m/z = 792.90–793.90, RT = 4.68 min).

Figure 5

Cluster analysis of HhUGT74AG11 and UGTs of other plants. Different UGT subfamilies are encoded with diverse colors. The plant species and the accession numbers of the input sequences are provided in Table S4.