Enzymatic preparation of 1-O-hydroxycinnamoyl-β-D-glucoses and their application to the study of 1-O-hydroxycinnamoyl-β-D-glucose-dependent acyltransferase in anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis

Abstract

Four 1-O-hydroxycinnamoyl-β-D-glucoses (HCA-Glcs), sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-glucoses, were synthesized using a recombinant protein of sinapate glucosyltransferase from Gomphrena globosa coupled with a recycling system of UDP-glucose by sucrose synthase from Arabidopsis thaliana. The substrate preference of HCA-Glc-dependent acyltransferase activity was examined in a protein extract prepared from anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis. The main anthocyanin molecule of the aglycon and the sugar moiety produced and accumulated in both cultured cells were exactly the same; the only difference was found in modification with sinapoyl moiety in D. carota and with feruloyl moiety in G. littoralis. The protein extracts from both D. carota and G. littoralis cultured cells showed higher activity with feruloyl-Glc than with sinapoyl-Glc. The major HCA-Glcs that accumulated in cultured cells of D. carota and G. littoralis were sinapoyl-Glc and feruloyl-Glc, respectively. These results suggested that the specificity of HCA moieties of major anthocyanin molecules in cultured cells of D. carota and G. littoralis might be dominated by produced and accumulated acyl donor molecules in vivo rather than by the substrate specificity of acyltransferase enzymes.