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 Arabidopsis thaliana O-methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1mm N-acetylserotonin (NAS). Among the eight strains harboring the full-length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A.thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The Km and Vmax values for ASMT activity were 233μm and 1800pmol/min/mg protein, while the Km and Vmax values for COMT activity were 103μm and 564,000pmol/min/mg protein, respectively. The catalytic efficiency (Vmax /Km ) 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 Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1mm NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.