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

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), w hich catalyzes the formation of scopolin from scopoletin, was purified appr oximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotian a tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to ap parent 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 enz yme did not require metal cofactors for activity. Its activity was inhibite d by Zn2+, Co2+ and Cu2+ ions, as well as by SH-blocking reagents. The K-m values for UDP-glucose, scopoletin and esculetin were 43, 150 and 25 mu M. respectively. A study of the initial rate of the reaction suggested that th e reaction proceeded via a sequential mechanism. The purified enzyme prefer red hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequ ence of CGTase revealed strong homology to the amino acid sequences of othe r glucosyltransferases in plants. (C) 2000 Elsevier Science Ireland Ltd. Al l rights reserved.