Biochemical and structural characterization of a guaiacol O-methyltransferase from Magnolia salicifolia

Volatile benzenoids such as veratrole and methyleugenol, together with terpenoids, constitute the characteristic floral scent of anise magnolia (Magnolia salicifolia), and the composition of these volatiles varies among natural populations in Japan. However, the enzymes responsible for biosynthesis of these volatile benzenoids remain poorly understood. In this study, we identified and biochemically characterized an S-adenosyl-l-methionine-dependent O-methyltransferase (MsOMT) from M. salicifolia. Recombinant MsOMT catalyzed methylation of guaiacol and eugenol to produce veratrole and methyleugenol, respectively. Kinetic analysis revealed apparent K_m values of 18 μM for guaiacol and 16 μM for eugenol. In contrast, the enzyme showed only weak activity toward bulkier phenolic substrates such as isorhapontigenin and isorhamnetin, despite their structural similarity to guaiacol-derived compounds. Homology modeling of MsOMT supported these biochemical findings and suggested that a compact substrate-binding pocket underlies the enzyme's preference for small phenolic substrates. These results provide the first biochemical characterization of a guaiacol O-methyltransferase from M. salicifolia and provide new insights into the structural basis of substrate recognition in plant O-methyltransferases.