The structurally homologous methyltransferases DnrK and RdmB catalyze mechanistically distinct and chemically atypical reactions during anthracycline biosynthesis. Through comprehensive functional analysis, we identified glutamic acid 299 as the critical molecular determinant responsible for their divergent catalytic behaviors. The substitution of E299 with a hydrophobic residue in DnrK was sufficient to confer RdmB-like hydroxylation activity, while the reverse mutation in RdmB introduced the decarboxylation capability. By systematically substituting E299 with residues of different properties, we successfully engineered a hybrid variant that integrates the functions of both parental enzymes capable of simultaneously producing four distinct types of anthracycline derivatives. These findings provide mechanistic insights into how subtle active-site modifications can drive the functional diversification of enzymes during evolution. Compounds 2 and 4 exhibited cytotoxicity against K-562 cells at the nanomolar level, demonstrating approximately 15-fold and 20-fold greater potency, respectively, compared to doxorubicin. This enhancement in antiproliferative activity underscores how strategic structural diversification of the anthracycline scaffold can improve pharmacological properties. Collectively, by elucidating the evolutionary strategy of DnrK and RdmB, our work provides potential next-generation anthracycline derivatives with optimized therapeutic profiles.