Isolation and Characterization of a UDPGlucose: Flavonol O³-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings

Abstract

A UDPGlc:flavonol 03-glucosyltransferase (EC 2.4.1.91) that catalyzes the formation of quercetin and kaempferol 03-glucosides has been purified about 1450-fold from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings with a 3.3% yield. Purification of the enzyme was achieved by (NH4)2SO4-precipitation, gel-filtration, ion-exchange chromatography on DEAE-Bio-Gel and Q-Sepharose, chromatofocusing, and electrophoresis in nondenaturing polyacrylamide (10%) gels. The enzyme preparation had a pH opfimum between 5.8 and 6.2, isoelectric point in the pH range 4.25 to 4.55, a M, of 59,000, and it was composed of two similar subunits of M, 29,500. The glucosyltransferase reached half substrate saturation at 180 micromolar (UDPGlc) and 7 micromolar (quercetin) concentrations. Kaempferol, which was glucosylated at a relative rate of 87%, had a lesser affinity for the enzyme (Km -12 micromolar). Flavanones, flavanols, flavones, dihydroflavonols, and anthocyanidins were not readily utilized as substrates, suggesting that the enzyme is specific for flavonol glucoside biosynthesis.Glycosylation of flavonoid compounds is thought to occur at or near the end of the biosynthetic pathway (3). The reaction renders a water-soluble product that can be transported to the vacuole, where in general flavonoid glycosides accumulate (9). The most commonly found sugar residue of flavonoid compounds is glucose, located in the 3-position of the molecule, that is transferred from UDPGlc2 to the flavonoid acceptor by the enzyme UDPGlc:flavonoid 03-glucosyltransferase (24).Illuminated red cabbage seedlings synthesize eight anthocyanins (6) that are the major flavonoid constituents of this plant. These are the 3-sophoroside-5-glucoside of cyanidin, and its acylated derivatives with malonic, p-coumaric, ferulic, and sinapic acids (6). While anthocyanins are the most investigated flavonoid components in the Brassicaceae, the presence of flavonol glycosides was also reported (4). Newer reports have confirmed quercetin (5) and kaempferol (23) glycosides as the most commonly occurring flavonol derivatives. Although activation ofenzymes in the phenylpropanoid ' Supported in part by Cornell University and Hatch funds. 2 Abbreviations: UDPGlc, uridine diphosphate glucose; 2-ME, 2-mercaptoethanol; PBE, polybuffer exchanger; PPO, diphenyloxazole; KPi, potassium phosphate. and specific anthocyanin pathway and end-product formation in Brassica was previously investigated in our laboratory, not much is known about the simultaneous synthesis and accumulation of flavonol glycosides.In the metabolism of these compounds, the first glycosylating step is the introduction of glucose into the 3-position of the molecule. Subsequent glucosylations of the introduced sugar and of the hydroxyl group in the 5-position give rise to the 3-sophoroside-5-glucoside of cyanidin and possibly the 3-sophorosides of quercetin and kaempferol.An enzyme that is responsible for glucosylating cyanidin in red cabbage seedlings was reported earlier and purified 50-fol...

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells

“…Isoelectric focusing indicated that the CGTase had a pI of 5.0. Other plant GTases associated 11 with secondary metabolism also have molecular masses of 40-62 kDa [17], consist of a single subunit or are dimers of subunits of 20-32 kDa, and have a pI near 5.0 [5,7,[19][20][21]. Thus, the physical characteristics of the CGTase were similar to those of other plant GTases.…”

Cloning and molecular characterization of R2R3-MYB and bHLH-MYC transcription factors from Citrus sinensis

“…For example, quercetin glycosides, such as rutin (quercetin 3-rutinoside), have a cyanidin glycosides-like structure and are among the main flavonoids in citrus leaves (Kawaii et al 2000). UFGTs from red cabbage seedlings and apple are able to use quercetin more efficiently than cyanidin as a substrate (Sun and Hrazdina 1991;Ju et al 1995). Recently, Offen et al (2006) showed that red grape UFGT (VvGT1), responsible for the formation of anthocyanins, is active, in vitro, on a range of flavonoids.…”

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

“…The ability to separate S-GT from this widely occurring flavonol glucosyltransferase activity (Sun and Hrazdina, 1991) provides further evidence that S-GT is specific for thiohydroximates. The enzyme does not act on oxygen analogs of hydroximates (Reed et al, 1993).…”