Molecular cloning and characterization of caffeic acid O-methyltransferase from Acorus calamus.

Volatile phenylpropenes, including α- and β-asarone, are characteristic aromas of sweet flag (Acorus calamus) and have been used as ingredients in pharmaceutical applications. However, studies on the biosynthetic enzymes involved in the production of volatile phenylpropenes in A. calamus remain limited. In this study, we analyzed volatile phenylpropenes, including α- and β-asarone, in the A. calamus plants. Using RNA-sequencing analysis, we identified a gene encoding S-adenosyl-L-methionine-dependent O-methyltransferase (AcCOMT), which converts caffeic acid to ferulic acid via the methylation of the meta-hydroxy group. The recombinant AcCOMT protein expressed in Escherichia coli specifically catalyzed O-methylation of the meta-hydroxy group of caffeic acid, 5-hydroxyferulic acid, and 5-hydroxyconiferyl alcohol. In contrast, it exhibited no detectable activity toward catechol-type stilbenes and flavonoids, such as piceatannol and quercetin. Additionally, no activity was observed towards the putative precursors of α-asarone and β-asarone, 6-hydroxy-(E)-isoeugenol and 6-hydroxy-(Z)-isoeugenol, respectively. Phylogenetic analysis revealed that AcCOMT has a distant evolutionary relationship to canonical COMT proteins from other plant species. Our results suggest that AcCOMT enzymes diverged early and followed a unique evolutionary trajectory, distinct from that of other COMTs, rather than originating through convergent evolution.