Yuzhuang Fu, Kejin Zhu, Fuwen Liang, Wenli Wang, Shanyong Yi, Huihui Zeng, Xingchen Lu, Libo Zhang, Jiaxin He, Wanqiu Liu, Xiaojing Ma, Dingqiao Xu, Bangxing Han, Jian Xu, Yucheng Zhao
Despite the broad reactivity of Fe(II)/2-oxoglutarate-dependent (Fe/2OG) enzymes, the basis for achieving selective O -demethylation remains a challenge. Levo -corydalmine ( S -CDL), a rare benzylisoquinoline alkaloid with potent non-opioid analgesic activity comparable to morphine, exemplifies this challenge. Here, we uncover a counterintuitive biosynthetic strategy in Corydalis yanhusuo involving early methylation followed by selective oxidative demethylation. Building on this hypothesis, we identify and engineer two Fe/2OG oxygenases, ODM4 and ODM5, capable of O -demethylating ( S )-tetrahydropalmatine ( S -THP), yet exhibiting distinct regioselective preferences. ODM4 favors C-10 O -demethylation to produce S -CDL as the major product, whereas ODM5 is functionally specialized for C-2 O -demethylation, leading exclusively to the formation of ( S )-tetrahydrojatrorrhizine ( S -THJ). Structural and computational analyses suggest that these preferences are dictated by substrate binding orientation and active site conformational constraints, with Leu113, Arg201, and Val225 as key determinants. Targeted mutagenesis of ODM4 yielded the M2 (L113A/R201A) variant that exhibits improved catalytic efficiency toward S -CDL formation while minimized off-pathway activity. Engineered ODM5 variants similarly acquired S -CDL activity absent in the wild type. These findings elucidate the molecular basis of selective O -demethylation and establish a design strategy for engineering selective oxidative tailoring enzymes in alkaloid biosynthesis.