Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.
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Five glucosyltransferases were cloned by RT-PCR amplification using total RNA from Hieracium pilosella L. (Asteraceae) inflorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially purified and functionally characterised as UDP-glucose:flavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed different regiospecificity profiles. UGT95A1 established a new subgroup within the UGT family showing high regiospecificity towards the C-3′ hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4′-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this flavones glucoside in H. pilosella flower heads. Semi quantitative expression profiles revealed that UGT95A1 was expressed at all stages of inflorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to flower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards flavones and flavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolites.
KeywordsAsteraceae Flavonoids Glucosyltransferase Hieracium Regiospecificity Substrate specificity
Plant secondary product glycosyltransferases
Rapid amplification of cDNA ends
High-speed counter current chromatography
This work was supported by the Deutsche Forschungsgemeinschaft and Dr. Sofia Moco is financed by the EU project “META-PHOR” (FOOD-CT-2006-036220).
- Arts ICW, Hollman PCH (2005) Polyphenols and disease risk in epidemiologic studies. Am J Clin Nutr 81 suppl:317S–325SGoogle Scholar
- Dombrowicz E, Świątek L, Kopycki W (1992) Phenolic acids in Helichrysi inflorescentia and Herba Hieracii pilosellae. Pharmazie 47:469–470Google Scholar
- Fukuchi-Mizutani M, Okuhara H, Fukui Y, Nakao M, Katsumoto Y, Yonekura-Sakakibara K, Kusumi T, Hase T, Tanaka Y (2003) Biochemical and molecular characterization of a novel UDP-glucose:anthocyanin 3′-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from Gentian. Plant Physiol 132:1652–1663PubMedCrossRefGoogle Scholar
- Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Bélanger A, Fournel-Gigleux S, Green M, Hum DW, Iyanagi T, Lancet D, Louisot P, Magdalou J, Chowdhury JR, Ritter JK, Schachter H, Tephly TR, Tipton KF, Nebert DW (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7:255–269PubMedCrossRefGoogle Scholar
- Noguchi A, Saito A, Homma Y, Nakao M, Sasaki N, Nishino T, Takahashi S, Nakayama T (2007) A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (Glycine max) seedlings purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis. J Biol Chem 282:23581–23590PubMedCrossRefGoogle Scholar
- Sawada S, Suzuki H, Ichimaida F, Yamaguchi M, Iwashita T, Fukui Y, Hemmi H, Nishino T, Nakayama T (2005) UDP-glucuronic acid:anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers: enzymology and phylogenetics of a novel glucuronosyltransferase involved in flower pigment biosynthesis. J Biol Chem 280:899–906PubMedCrossRefGoogle Scholar