Transformation of acridone synthase to chalcone synthase

Acridone synthase (ACS) and chalcone synthase (CHS) catalyse the pivotal re actions in the formation of acridone alkaloids or flavonoids. While acridon e alkaloids are confined almost exclusively to the Rutaceae, flavonoids occ ur abundantly in all seed-bearing plants. ACSs and CHSs had been cloned fro m Ruta graveolens and shown to be closely related polyketide synthases whic h use N-methylanthraniloyl-CoA and 4-coumaroyl-CoA, respectively, as the st arter substrate to produce the acridone or naringenin chalcone. As proposed for the related 2-pyrone synthase from Gerbera, the differential substrate specificities of ACS and CHS might be attributed to the relative volume of the active site cavities. The primary sequences as well as the immunologic al cross reactivities and molecular modeling studies suggested an almost id entical spatial structure for ACS and CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133 and Val265 were assumed to play a critical role in substrate specificity. Exchange of a single amino acid (Val265Phe) reduced the catalytic activity by about 75% but grossly shifted the specificity to wards CHS activity, and site-directed mutagenesis replacing all three resid ues by the corresponding amino acids present in CHS (Ser132Thr, Ala133-Ser and Val265Phe) fully transformed the enzyme to a functional CHS with compar atively marginal ACS activity. The results suggested that ACS divergently h as evolved from CHS by very few amino acid exchanges, and it remains to be established why this route of functional diversity bas developed in the Rut aceae only. (C) 2001 Federation of European Biochemical Societies. Publishe d by Elsevier Science B.V. All rights reserved.