Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl-CoAs with N-methylanthrani loyl-CoA or 4-coumaroyl-CoA to produce acridone alkaloids and flavonoid pig ments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chal cone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding r egions of these three cDNAs differ only marginally, and the translated poly peptides show about 90% identity with the CHSs from Citrus sinensis but les s than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4-Coumaroyl-CoA was the preferred starter subst rate for CHS1, but cinnamoyl-CoA and caffeoyl-CoA were also turned over at significant rates. However, N-methylanthraniloyl-CoA was not accepted. In c ontrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4-coumaroyl-CoA, which on ave rage reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined w ith N-methylanthraniloyl-CoA as the starter substrate, while the conversion of cinnamoyl-CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone format ion. Nevertheless, our results suggest that very minor changes in the seque nces of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site-directed mut agenesis.