In vitro inhibition of carrot chalcone synthase by 3'-nucleotidase: the role of the 3'-phosphate group of malonyl-coenzyme A in flavonoid biosynthesis.

In mixing experiments with extracts derived from two cell lines of Daucus carota tissue cultures with and without chalcone synthase activity, strong inhibition of chalcone synthase (CHS) became obvious. This inhibition was due to the presence of a heatlabile protein in extracts from cells devoid of CHS activity. This protein was partially purified and identified as 3'-nucleotidase (EC 3.1.3.6). Inhibition was also observed in the presence of purified 3'-nucleotidase from Lolium multiflorum. The phosphate group in the 3'-position of adenosine, a part of the CoA thioester substrates of CHS, was hydrolyzed by this enzyme. The dephosphorylated form of malonyl-CoA was no longer a substrate, whereas 4-coumaryl-3'-dephospho-CoA as well as 4-coumaryl-CoA was still able to act as a primer for the CHS reaction. Further studies showed that malonyl-3'-dephospho-CoA was an efficient CHS inhibitor. On the other hand, CoA-SH lost its inhibitory activity after dephosphorylation in the 3'-position. These results are discussed with respect to the mechanism of chalcone synthesis.