Molecular and Structural Characterization of a Chalcone di-<i>C</i>-Methyltransferase RdCMT from <i>Rhododendron dauricum</i> and Its Application in De Novo Biosynthesis of Farrerol in <i>Pichia pastoris</i>
Meng Zhang, Yang‐Oujie Bao, Zonglin Dai, Zhilan Qian, Haishuang Yu, Jia-Jing Zhou, Yi Chen, Zilong Wang, Kaituo Wang, Menghao Cai, Min Ye
Abstract
Methylation plays a crucial role in drug design and optimization. While numerous methyltransferases have been characterized from plants, C -methyltransferases, particularly those targeting phenolic skeletons, are rare. In this study, we identified a novel di- C -methyltransferase RdCMT from the medicinal plant Rhododendron dauricum . RdCMT catalyzes a sequential two-step 3′- C /5′- C -methylation of naringenin chalcone, leading to the biosynthesis of farrerol. RdCMT exhibited a strict substrate specificity for chalcones. Through combinatorial catalysis, a series of C -methylated flavonoids were synthesized. Moreover, farrerol was synthesized de novo in Nicotiana benthamiana and Pichia pastoris with yields of 0.4 mg/g (dry weight) and 149.0 mg/L, respectively. The structure of RdCMT was determined using cryo-electron microscopy (cryo-EM), revealing that residues R328 and G296 significantly influence the substrate specificity of RdCMT. This work not only introduces a potent biocatalyst for the preparation of C -methylated flavonoids but also offers insights into the catalytic mechanisms of C -methyltransferases.