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

Abstract

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1,200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%.Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn 2+ , Co 2+ and Cu 2+ ions, as well as by SH-blocking reagents. The Km values for UDP-glucose, scopoletin and esculetin were 43 µM, 150 µM and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

Exogenously added naphthols induce three glucosyltransferases, and are accumulated as glucosides in tobacco cells

“…The samples for enzyme assay were extracted from the residual cells with Tris-HCl (pH 7.5) containing 5 mM β-mercaptoethanol as described [28]. GTase reactions were performed for 60min at 30 ºC using 200 µM 1-or 2-naphthol and 500 µM UDP-glucose as substrates, then the generated glucoside was determined by HPLC.…”

Cloning and characterization of a glucosyltransferase that reacts on 7-hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco cells

“…Moreover, several GTase genes have been reported, namely, genes of IEGT, TOGT, jasmonate-inducible GTase gene (JIGT) [19], and salicylic acid GTase gene [20]. In our previous studies, the glucosylation activity in cultured tobacco cells was investigated [21][22][23]. We isolated GTase genes (NtGT1a, NtGT1b and NtGT3) whose encoding enzymes have glucosylation activity against many kinds of phenolics, such as flavonoids, coumarins and naphthols [22,23].…”

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

“…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.…”

“…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.…”