Abstract
We have previously identified the gene encoding 1-O-acylglucose:anthocyanin 3'-O-glucoside-6'''-O-acyltransferase (3'AT; CtAT1) which is responsible for the biosynthesis of ternatin C3 from ternatin C5 in blue butterfly pea flowers. This enzyme, consisted of heteromeric 31- and 24-kDa subunits, is a serine carboxypeptidase-like acyltransferase containing three potential N-glycosylation sites. Recombinant CtAT1 protein was expressed in Sf9 cells in a baculovirus expression vector. LC/MS analysis of the recombinant-enzymatic reaction mixtures, in which various ternatins were reacted as substrates, indicated that CtAT1 had antocyanin 5'-O-glucoside-acyltransferase (5'AT) and anthocyanin 3'-O-(4-O-glucosyl-(6-O-p-coumaroyl))glucoside-acyltransferase (3'AT2) activities as well as first 3'AT activity. To investigate the subcellular localization of CtAT1, fusion proteins of CtAT1-green fluorescent protein (GFP) were engineered and stably expressed in tobocco BY-2 cells. Expression of CtAT1-GFP fusion protein showed that CtAT1 localized to the vacuolar lumen. These results suggested that CtAT1 catalysed the acylation involved in polyacylated anthocyanin biosynthesis in the vacuole.
