Caffeic acid O‐methyltransferase is involved in the synthesis of melatonin by methylating N‐acetylserotonin in Arabidopsis

Abstract Although a plant N ‐acetylserotonin methyltransferase ( ASMT ) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight A rabidopsis thaliana O ‐methyltransferase ( OMT ) cDNA s in E scherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 m m N ‐acetylserotonin ( NAS ). Among the eight strains harboring the full‐length c DNA s, the OMT 3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT . OMT 3 is already known as caffeic acid OMT ( COMT ), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (At COMT ) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The K m and V max values for ASMT activity were 233 μ m and 1800 pmol/min/mg protein, while the K m and V max values for COMT activity were 103 μ m and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency ( V max / K m ) for ASMT activity was 709‐fold lower than for COMT . In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose‐dependent manner, but the activity of COMT was not altered by NAS . Lastly, the A rabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when A rabidopsis leaves were infiltrated with 1 m m NAS , suggestive of in vivo role of COMT in melatonin biosynthesis in plants.