Journal
JOURNAL OF PINEAL RESEARCH
Volume 57, Issue 2, Pages 219-227Publisher
WILEY
DOI: 10.1111/jpi.12160
Keywords
Arabidopsis; caffeic acid O-methyltransferase; melatonin; N-acetylserotonin methyltransferase; O-methyltransferase
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Funding
- Next-Generation BioGreen 21 Program (SSAC), Rural Development Administration [PJ00949105]
- Basic Research Program through National Research Foundation of the Ministry of Education, Science, and Technology (MEST), Republic of Korea [2010-0020141]
- National Research Foundation of Korea [22A20130012880, 2010-0020141] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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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 1 mM 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 K-m and V-max values for ASMT activity were 233 mu M and 1800 pmol/min/mg protein, while the K-m and V-max values for COMT activity were 103 mu 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 Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mM NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.
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