HYDROXYCINNAMIC ACID TRANSFERASES IN THE BIOSYNTHESIS OF ACYLATED BETACYANINS - PURIFICATION AND CHARACTERIZATION FROM CELL-CULTURES OF CHENOPODIUM-RUBRUM AND OCCURRENCE IN SOME OTHER MEMBERS OF THE CARYOPHYLLALES

An acyltransferase from cell cultures of Chenopodium rubrum was purified 515-fold with a 2.5 % recovery. This enzyme catalyzes the transfer of hydroxycinnamic acids from 1-0-hydroxycinnamoyl-beta-glucose to the C-2 hydroxy group of glucuronic acid of amaranthin (betanidin 5-O-glucuronosylglucose). The in vivo products formed are celosianin I (4-coumaroylamaranthin) and celosianin II (feruloylamaranthin). The enzyme can be classified as 1-0-hydroxycinnamoyl-beta-glucose: amaranthin O-hydroxycinnamoyl-transferase (EC 2.3. 1. -). Its molecular weight was determined by gel filtration column chromatography to be ca. 69.5 kDa. Maximal rate of product formation was found to be at pH 5.6. The isoelectric point of the enzyme was at pH 4.7. The reaction temperature maximum was at 37-degrees-C and the apparent energy of activation was calculated to be 44.5 kJ . mol-1. The enzyme showed a V(max) Of 9 1 0 pkat . (mg protein) - 1 with amaranthin as acceptor and feruloylglucose as acyl donor. The ratios of V(max)/K(m) values for sinapoyl-, feruloyl, caffeoyl- and 4-coumaroylglucoses were found to be 100 : 56 : 56 : 40. Donor competition experiments support the conclusion that one single enzyme is responsible for the ester formation with the different hydroxycinnamic acids. From the possible acceptors tested, only amaranthin (15S configuration) and isoamaranthin (15R) were esterified with K(m) values of 280 and 800-mu-M, respectively. Catalytic effectivity (V(max)/K(m) was found at a relative ratio 15S: 15R of 100 : 42. Betanin (betanidin 5-O-glucoside) and gomphrenin I (betanidin 6-O-glucoside) were not accepted. Some other acylated betacyanin-containing members of four families of the Caryophyllales were investigated and showed the same type of hydroxycinnamoyltransferase activity with 1-O-hydroxycinnamoylglucose as acyl donor, but with different acceptor molecule specificities.