title: Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens.
abstract: The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl-CoAs with N-methylanthraniloyl-CoA or 4-coumaroyl-CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less 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 substrate for CHS1, but cinnamoyl-CoA and caffeoyl-CoA were also turned over at significant rates. However, N-methylanthraniloyl-CoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4-coumaroyl-CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with 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 formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site-directed mutagenesis.
authors.affiliation: Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Springob
authors.forename: R
authors.initials: R
authors.lastname: Lukacin
authors.forename: C
authors.initials: C
authors.lastname: Ernwein
authors.forename: I
authors.initials: I
authors.lastname: Gröning
authors.forename: U
authors.initials: U
authors.lastname: Matern
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D002957:Citrus / Q000201:enzymology; D003001:Cloning, Molecular; D017343:Genes, Plant; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019685:Rosales / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D000217:Acyltransferases; C086702:acridone synthase; C020141:flavanone synthetase