Glycosylation plays an important role in the structural diversification of plant natural products.Identifying efficient glycosyltransferases is also a key step in synthesizing valuable glycosides.This research successfully isolated a glycosyltransferase gene from Lagotis brevituba Maxim, designated as UGT74DU1.UGT74DU1 comprises 1 413 base pairs, encoding a protein of 470 amino acid residues, with a calculated molecular mass of approximately 52.6 kDa.Bioinformatics analysis predicted an isoelectric point of 5.53.Phylogenetic and sequence alignment analyses revealed that UGT74DU1 shares the closest evolutionary relationship with CrUGT74AN3, despite exhibiting only 46.86% sequence similarity.UGT74DU1 was successfully expressed in Escherichia coli as soluble protein with the help of the chaperone plasmid pGro7.Subsequent functional characterization was performed through in vitro enzymatic assays.Through LC-MS analysis and NMR structural characterization, it was discovered that UGT74DU1 preferentially recognizes flavonoids such as quercetin, kaempferol, rutin, and isoquercitrin, catalyzing glucosylation at the C-7-OH position to produce flavonoid-7-O-glucosides.Furthermore, it could subsequently recognize the C-4′-OH position to generate flavonoid-4′,7-O-diglucosides.Moreover, it could catalyze the glycosylation of phenylethanoid glycosides such as verbascoside, demonstrating a certain degree of substrate promiscuity.
[1] JONES P, VOGT T.Glycosyltransferases in secondary plant metabolism:Tranquilizers and stimulant controllers[J].Planta, 2001, 213(2):164-174.
[2] WANG Z, WANG D, YANG D, et al.The effect of icariin on bone metabolism and its potential clinical application[J].Osteoporosis International, 2018, 29(3):535-544.
[3] GUO Q H, SHI M K, SARENGAOWA, et al.Recent advances in the distribution, chemical composition, health benefits, and application of the fruit of Siraitia grosvenorii[J].Foods, 2024, 13(14):2278.
[4] MISHRA P, AHSAN F, MAHMOOD T, et al.Progress of arbutin from dietary supplement to advanced medicine[J].eFood, 2024, 5(6):e70013.
[5] WANG Z L, WANG S, XU Z,et al.Highly promiscuous flavonoid 3-O-glycosyltransferase from Scutellaria baicalensis[J].Organic Letters, 2019, 21(7):2241-2245.
[6] YANG Y H, WU Y N, ZHUANG Y B, et al.Discovery of glycosyltransferases involved in the biosynthesis of ligupurpuroside B[J].Organic Letters, 2021, 23(20):7851-7854.
[7] JIAN X, SUN Q Y, XU W T, et al.Engineering the substrate specificity of UDP-glycosyltransferases for synthesizing triterpenoid glycosides with a linear trisaccharide as aided by ancestral sequence reconstruction[J].Angewandte Chemie (International Ed. in English), 2024, 63(48):e202409867.
[8] 中国科学院西北高原生物研究所. 藏药志[M].西宁:青海人民出版社, 1991:447.
Northwest Institute of Plateau Biology, Chinese Academy of Sciences.Tibetan Medicine Record[M].Xining:Qinghai People’s Publishing House, 1991:447.
[9] YUAN X, WEN H X, CUI Y L, et al.Phenolics from Lagotis brevituba Maxim[J].Natural Product Research, 2017, 31(3):362-366.
[10] YANG B L, HOU Q, HU F, et al.Total flavones derived from Lagotis brevituba Maxim reduce the levels of inflammatory cytokines in cerebral cortex and hippocampus of Alzheimer’s disease mice[J].Chinese Journal of Cellular & Molecular Immunology, 2016, 32(7):881-885.
[11] ROSS J, LI Y, LIM E, et al.Higher plant glycosyltransferases[J].Genome Biology, 2001, 2(2):REVIEWS3004.
[12] HUANG W, ZHANG X L, LI J H, et al.Substrate promiscuity, crystal structure, and application of a plant UDP-glycosyltransferase UGT74AN3[J].ACS Catalysis, 2024, 14(1):475-488.
[13] DAI X L, SHI X X, YANG C L, et al.Two UDP-glycosyltransferases catalyze the biosynthesis of bitter flavonoid 7-O-neohesperidoside through sequential glycosylation in tea plants[J].Journal of Agricultural and Food Chemistry, 2022, 70(7):2354-2365.
[14] CHIU H H, HSIEH Y C, CHEN Y H, et al.Three important amino acids control the regioselectivity of flavonoid glucosidation in glycosyltransferase-1 from Bacillus cereus[J].Applied Microbiology and Biotechnology, 2016, 100(19):8411-8424.
[15] YIN Q G, SHEN G A, DI S K, et al.Genome-wide identification and functional characterization of UDP-glucosyltransferase genes involved in flavonoid biosynthesis in Glycine max[J].Plant and Cell Physiology, 2017, 58(9):1558-1572.
[16] LIM C E, AHN J H, LIM J.Molecular genetic analysis of tandemly located glycosyltransferase genes UGT73B1 UGT73B2, and UGT73B3, in Arabidopsis thaliana[J].Journal of Plant Biology, 2006, 49(4):309-314.
[17] OKITSU N, MATSUI K, HORIKAWA M, et al.Identification and characterization of novel Nemophila menziesii flavone glucosyltransferases that catalyze biosynthesis of flavone 7,4′-O-diglucoside, a key component of the blue metalloanthocyanins[J].Plant and Cell Physiology, 2018, 59(10):2075-2085.
[18] KIM B G, SUNG S H, AHN J H.Biological synthesis of quercetin 3-O-N-acetylglucosamine conjugate using engineered Escherichia coli expressing UGT78D2[J].Applied Microbiology and Biotechnology, 2012, 93(6):2447-2453.
[19] LIAO B, LIU X J, LI Y J, et al.Functional characterization of a highly efficient UDP-glucosyltransferase CitUGT72AZ4 involved in the biosynthesis of flavonoid glycosides in Citrus[J].Journal of Agricultural and Food Chemistry, 2025, 73(9):5450-5464.
[20] WANG Z L, DU X Q, YE G, et al.Functional characterization, structural basis, and protein engineering of a rare flavonoid 2′-O-glycosyltransferase from Scutellaria baicalensis[J].Acta Pharmaceutica Sinica B, 2024, 14(8):3746-3759.
[21] BONACCORSI P, CARISTI C, GARGIULLI C, et al.Flavonol glucoside profile of southern Italian red onion (Allium cepa L.)[J].Journal of Agricultural and Food Chemistry, 2005, 53(7):2733-2740.
[22] LI C, ROY J K, PARK K C, et al.pH-promoted O-α-glucosylation of flavonoids using an engineered α-glucosidase mutant[J].Bioorganic Chemistry, 2021, 107:104581.
[23] YAN Y R, MO T, HUANG W Q, et al.Glycosylation of aromatic glycosides by a promiscuous glycosyltransferase UGT71BD1 from Cistanche tubulosa[J].Journal of Natural Products, 2022, 85(7):1826-1836.
[24] WANG Z L, WEI W Q, WANG H D, et al.Functional characterization, structural basis, and regio-selectivity control of a promiscuous flavonoid 7,4′-di-O-glycosyltransferase from Ziziphus jujuba var.spinosa[J].Chemical Science, 2023, 14(16):4418-4425.
