C-Prenylation of dihydrochalcones by prenyltransferases AnaPT and its engineered mutants

Prenylated dihydrochalcones mainly occur in plants and show various biological activities. In biological systems, the enzymatic introduction of prenyl groups is catalyzed by prenyltransferases. Notably, fungal prenyltransferases exhibit significant substrate promiscuity, accepting diverse aromatic acceptors and utilizing various prenyl donors. In this study, saturation mutagenesis at I266, a key residue in AnaPT((R)-benzodiazepindinone prenyltransferase from Neosartorya fischer) for dimethylallylating the dihydrochalcone phloretin (1), yielded 19 mutants. Among these, mutants AnaPT_I266A, AnaPT_I266T, and AnaPT_I266C exhibited enhanced selectivity toward 1 compared to the wild-type enzyme. Subsequently, the substrate scope of AnaPT and its mutants was evaluated using four additional dihydrochalcones in the presence of prenyl donors of varying chain lengths. Notably, the I266T mutant enzyme catalyzed the conversion of substrate 3 to a new compound, 3D1, which was not detected in reaction catalyzed by the wild-type enzyme. Compared to the wild-type enzyme, the I266A mutant exhibited a ten-fold increase in the yield of product 3D2. Furthermore, mutant I266T catalyzed the conversion of substrate 3 to product 3G with altered prenyl donor specificity- a reaction that yielded no detectable products with wild-type AnaPT. This enzymatic screening yielded eight prenylated dihydrochalcones. Among these, seven compounds (2D1, 2D2, 3D1, 3D2, 4D1, 2G, and 3G) represent new structures unreported prior to this study. Structural elucidation confirmed that all products are exclusively C-prenylated dihydrochalcones, demonstrating the strict chemoselectivity of AnaPT and its engineered variants.