Structural insights into a dual-substrate O-methyltransferase reveal the enzymatic mechanism of parallel paeonol biosynthesis.

Paeonol, the medicinal indicator component of the traditional Chinese medicine 'Mudanpi', has an incompletely elucidated biosynthetic pathway, particularly its post-modification steps. Specifically, the post-modification processes involve 4'-O-methylation and 2'-hydroxylation, thus the formation can occur either sequentially or in parallel. Here, using integrated transcriptomic and metabolomic analyses, we identify a key dual-substrate 4'-O-methyltransferase, PoOMT1, in Paeonia ostii. In vitro assays demonstrate that PoOMT1 exhibits high affinity, catalytic efficiency, and strict regioselectivity toward two paeonol precursors: 4'-hydroxyacetophenone and 2',4'-dihydroxyacetophenone. Heterologous expression in tobacco and silencing in P. ostii of PoOMT1 further confirm its role in paeonol biosynthesis, supporting a parallel pathway. Structural analysis of the PoOMT1 ternary complex, combined with mutagenesis studies, uncover a His-Asn-Gln catalytic triad essential for the 4'-O-methylation, and reveal a T-shaped substrate binding pocket. This pocket operates through dual-anchor and steric constraint mechanisms, which provide the structural basis for strict regioselectivity toward the 4'-OH group and facilitate the parallel biosynthetic pathway. Moreover, the divergent catalytic profiles of ten PoOMT1 homologs that recognize the same substrates further corroborate the proposed catalytic mechanism. Our work elucidates the molecular basis of 4'-O-methylation and parallel biosynthetic pathway of paeonol, and lays a foundation for the complete elucidation of its pathway.