Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa

Kramer, Catherine; Prata, Rogerio T. N.; Willits, Michael G.; Luca, Vincenzo De; Steffens, John C.; Graser, Gerson

doi:10.1016/s0031-9422(03)00507-7

Cited by 80 publications

(64 citation statements)

References 24 publications

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“…The aYnity to umbelliferone, however, is rather common among UGTs, because out of 90 A. thaliana UGTs 48 were capable to glycosylate hydroxycoumarins (Lim et al 2003). Recent studies indicate that most plant UGTs are not absolutely substrate speciWc at least in vitro (Vogt and Jones 2000;Messner et al 2003;Kramer et al 2003;Lunkenbein et al 2006;Tian et al 2006;Suzuki et al 2007). The here described in vitro glucosylation pattern of H. pilosella UGTs could therefore not be attributed directly to in vivo functions.…”

Section: Discussionmentioning

confidence: 99%

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

Witte

Moco

Vervoort

et al. 2009

Planta

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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.

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Section: Discussionmentioning

confidence: 99%

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

Witte

Moco

Vervoort

et al. 2009

Planta

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“…The biochemical data available on UGTs enable some conclusions on UGT acceptor specificity to be drawn. Several UGTs have broad acceptor specificity as demonstrated by their ability to glycosylate a large number of structurally quite different aglycones in vitro (Vogt et al, 1997;Vogt and Jones, 2000;Hansen et al, 2003;Kramer et al, 2003;Modolo et al, 2007). Other UGTs are highly specific for a single acceptor (Fukuchi-Mizutani et al, 2003;Kramer et al, 2003).…”

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confidence: 99%

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

et al. 2008

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The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidinderived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the substrate specificity of isolated wild-type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 2.5% of wild-type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDPglucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild-type enzyme activity toward delphinidin-3-O-glucoside was only 5% to 10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B ring were introduced to increase the activity toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity.

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“…1A). As one of the major antioxidants derived from plant sources, quercetin is beneficial to human health and has been ascribed anticancer (20 -22), anti-inflammatory (23,24), and anti-allergic activity (25,26). In nature quercetin is often glycosylated on one or more of the five hydroxyl groups to increase its solubility, stability, and bioavailability.…”

mentioning

confidence: 99%

“…Recent studies have suggested that many plant secondary metabolite UGTs are more regiospecific than substrate specific (11,(22)(23)(24)(25)(26). Recently, UGTs from Arabidopsis thaliana were extensively analyzed both in vitro and in vivo with quercetin as acceptor; from a total of 29 UGTs that exhibited activity with quercetin, 14 catalyzed glycosylation either on the C-ring 3-hydroxyl or the A-ring 7-hydroxyl, the most commonly reported glycosylation positions for quercetin derivatives isolated from plants.…”

mentioning

confidence: 99%

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

Wang

Dixon

2006

Journal of Biological Chemistry

113

115

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Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa

Cited by 80 publications

References 24 publications

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

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

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

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