Purification and Characterization of UDP-Glucose : Curcumin Glucoside 1,6-Glucosyltransferase from Catharanthus roseus Cell Suspension Cultures

Catharanthus roseus cell suspension cultures converted exogenously added curcumin to a series of curcumin glucosides that possessed drastically enhanced water solubility. A cDNA clone encoding a glucosyltransferase responsible for glucosylation of curcumin to form curcumin 4′-O-glucoside was previously isolated, and in the present study a novel sugar–sugar glycosyltransferase, UDP-glucose:curcumin glucoside glucosyltransferase (UCGGT), was purified approximately 900-fold to apparent homogeneity from cultured cells of C. roseus. The purified enzyme (0.2% activity yield) catalyzed 1,6-glucosylation of curcumin 4′-O-glucoside to yield curcumin 4′-O-gentiobioside. The molecular weight and isoelectric point were estimated to be about 50 kDa and 5.2, respectively. The enzyme showed a pH optimum between 7.5 and 7.8. Both flavonoid 3-O– and 7-O-glucosides were also preferred acceptor substrates of the enzyme, whereas little activity was shown toward simple phenolic glucosides such as arbutin and glucovanillin, cyanogenic glucoside (prunasin) or flavonoid galactoside. These results suggest that UCGGT may also function in the biosynthesis of flavonoid glycosides in planta.

Here we describe the purification and characterization of the second enzyme, UDP-glucose:curcumin glucoside 1,6-glucosyltransferase, which we hereafter refer to as UCGGT. To our knowledge, this is the first report describing purification and characterization of a 1,6-glucosyltransferase acting upon phenolic glucosides.

Substrate specificity of UCGGT

Chemical structures of phenolic glycosides used as glucosyl acceptors to examine the substrate specificity of UCGGT.

The glucosyl acceptor specificity of UCGGT was examined using various phenolic glucosides. HPLC analysis revealed the formation of a product with higher water solubility than the substrate when flavonoid glucosides were used as acceptor substrates, whereas no such products were detected from other phenolic glucosides. A product peak detected when UCGGT was incubated with kaempferol 3-O-glucoside displayed a retention time and UV spectrum consistent with those of authentic kaempferol 3-O-gentiobioside. The products from quercetin 3-O-glucoside, myricetin 3-O-glucoside and luteolin 7-O-glucoside were also tentatively identified as their gentiobioside derivatives. Although these flavonoid metabolites could serve as UCGGT substrates, kinetic analysis revealed that Cmg was the most favorable acceptor substrate among those examined. Among the flavonoid derivatives tested, UCGGT exhibited appreciable activity toward kaempferol 3-O-glucoside and quercetin 3-O-glucoside, while the Km value and specific activity for luteolin 7-O-glucoside were almost 10-fold higher and 8-fold lower, respectively, than those for kaempferol 3-O-glucoside. UCGGT exhibited glycosyl transfer activity with UDP-galactose as a glycosyl donor substrate. Although the apparent Km for UDP-galactose was lower than that for UDP-glucose, the specific activity was only about a quarter of that for UDP-glucose. (Plant and Cell Physiology, Volume 48, Issue 11, November 2007, Pages 1635–1643)