Major anthocyanin biosynthesis in the brilliant crimson petals from Erythrina crista-galli L

Abstract

The anthocyanins in the brilliant crimson petals from Erythrina crista-galli L. were extracted with 10% AcOH, and the extract was purified by Amberlite XAD-7 column chromatography, paper chromatography, and preparative HPLC. The structure of a major anthocyanin was confirmed as cyanidin 3-O-sophoroside by 1H and 13C NMR and FABMS. Then, the catalysis of UDP-glucose: flavonoid 3-O-glucoside glucosyltransferase (3GGT) was predicted at the final step in the anthocyanin biosynthesis. In this study, the 3GGT was purified approximately to 800-fold from E. crista-galli petals by eight-steps purification procedures. The purified enzyme (Ec3GGT) catalyzed only the 2″-O-glucosylation of flavonoid 3-O-glucoside to produce flavonoid 3-O-sophoroside. Ec3GGT showed a pH optimum around 7.5. The molecular weight and isoelectric point were estimated to be about 82kDa and 6.5, respectively; the molecular weight was larger than known UDP-sugar: flavonoid glycosyltransferases (UFGTs). The enzyme did not require metal cofactors but inhibited by Zn2+, Co2+ and Cu2+, and it activity was inhibited by SH-blocking reagents such as NEM and PCMB. Both anthocyanidin 3-O-glucosides and flavonol 3-O-glucosides were favorable acceptor substrates of the enzyme, whereas no activity was shown toward C-glucosylflavones such as isovitexin, which is a minor component in the petals. These results showed that Ec3GGT involved in the last step of biosynthesis of a major anthocyanin in petals.