Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.

Abstract

Five glucosyltransferases were cloned by RT-PCR ampliWcation using total RNA from Hieracium pilosella L. (Asteraceae) inXorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially puriWed and functionally characterised as UDP-glucose:Xavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed diVerent regiospeciWcity proWles. UGT95A1 established a new subgroup within the UGT family showing high regiospeciWcity towards the C-3Ј hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4Ј-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this Xavones glucoside in H. pilosella Xower heads. Semi quantitative expression proWles revealed that UGT95A1 was expressed at all stages of inXorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to Xower 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 Xavones and Xavonols 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.

Identification and characterization of UDP-glucose:Phloretin 4′-O-glycosyltransferase from Malus x domestica Borkh

“…S6). It is somewhat unusual that three products were formed because usually glycosyltransferases are rather regioselective concerning the glycosylation site (Vogt et al, 1999;Isayenkova et al, 2006;Griesser et al, 2008;Witte et al, 2009;Gosch et al, 2012).…”

“…In general, glycosyltransferases are known as a supergene family with low sequence similarity and only few highly conserved regions (Griesser et al, 2008). They are described as predominantly regioselective or regiospecific concerning the sugar attachment site, but are usually not highly substrate-specific (Vogt and Jones, 2000;Treutter, 2001;Griesser et al, 2008;Witte et al, 2009). The glycosylated product of phloretin (5) and UDP-glucose comigrated with a known trilobatin (6) standard, indicating that MdPh-4 0 -OGT is likely to be an enzyme that glycosylates phloretin (5) to trilobatin (6) in planta.…”

Characterization of a Glucosyltransferase Enzyme Involved in the Formation of Kaempferol and Quercetin Sophorosides in Crocus sativus

“…Among the functionally characterized UGTs, UGT707B1 shares the highest identity (40%) with UGT88A4 from Maclura pomifera, which did not exhibit any activity toward flavonoids and isoflavonoids but catalyzed the glucosylation of coumarin susbtrates, although the in vivo substrate is unknown (Tian et al, 2006). The same identity is shown with UGT88A8 and UGT88A9 from Hieracium pilosella, which showed activity with flavones, flavonols, caffeic acid, esculetin, catechol, resorcinol, and hydroquinone (Witte et al, 2009). …”

Substrate specificity and contribution of the glycosyltransferase UGT71A15 to phloridzin biosynthesis

“…Three cDNA clones encoding glycosyltransferases were identified, but only one showed high specificity for phloretin in vitro (Gosch et al 2010b;Jugde et al 2008). The others accepted a broad range of flavonoid substrates to different extents and it was unclear if the ability of phloretin 2 0 -O-glucosylation was of physiological importance or exclusively owed to the general broad substrate specificity of the two enzymes as frequently observed for glucosyltransferases (Griesser et al 2008;Halbwirth et al 1997;Isayenkova et al 2006;Stich et al 1994Stich et al , 1997Treutter 2001;Witte et al 2009). To investigate if the moderate phloretin acceptance observed in vitro reflects an active participation in phloridzin biosynthesis in planta, we created transgenic apple plants overexpressing the UGT71A15 cDNA clone.…”

“…The positions of quercetin carrying deprotonated hydroxyl groups at the optimal pH correlate with the fact that two different products were formed in vitro, which could be identified as the respective 3-O-glucosides and the 7-O-glucosides. On the other hand, it was surprising that two products were formed because in general glycosyltransferases are rather regioselective concerning the glycosylation site (Griesser et al 2008;Isayenkova et al 2006;Treutter 2001;Witte et al 2009). The observed formation of flavonol 7-O-glucosides by UGT71A15 in vitro is probably not of physiological relevance, because flavonols 7-O-glucosides are not native in apple and flavonols 3-O-glucosides are exclusively formed when enzyme preparations from apple leaves replace the recombinant enzyme as protein source.…”