Cloning of Arabidopsis serotonin N‐acetyltransferase and its role with caffeic acid O‐methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations

Abstract

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis.

Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase

“…Dual expression of SNAT and COMT in E. coli resulted in the production of both Nacetylserotonin and melatonin, which promotes beneficial biological activities in animals and plants (Sompol et al 2011;Lee et al 2014b). Several highly relevant differences in the melatonin biosynthetic genes between animals and plants were identified through comparisons with the melatonin synthetic capacity in the heterologous E. coli system.…”

A new balancing act: The many roles of melatonin and serotonin in plant growth and development

“…Recently, a caffeic acid O-methyltransferase (COMT) was found to convert Nacetylserotonin to MEL in plants 32,37,41 . This suggests that alternate routes and enzymes may be involved in this process.…”

“…TDC in plants and SNAT in animals are generally considered to be the rate limiting steps in these pathways. For example transgenic studies in rice have found that up-regulation of TDC can increase endogenous 5HT and MEL Downloaded by [University of Leeds] at 08:27 04 October 2015 7concentrations; however limited literature has also suggested that ASMT may be a more important in determining the amount of melatonin produced in some plant species 34,39,40 .Recently, a caffeic acid O-methyltransferase (COMT) was found to convert Nacetylserotonin to MEL in plants 32,37,41 . This suggests that alternate routes and enzymes may be involved in this process.…”

Cloning and Phylogenetic Analysis of Brassica napus L. Caffeic Acid O-Methyltransferase 1 Gene Family and Its Expression Pattern under Drought Stress

“…A concomitant increase in the precursor 5-hydroxyguaiacyl units and a decrease in the S/G ratio are also frequently observed. Interestingly, a recent study implied that AtOMT methylates N-acetylserotonin into melatonin possessing N-acetylserotonin O-methyltransferase (ASMT) activity [15], results that were also evaluated and identified in rice [16]. Melatonin is implicated in pleiotropic functions during plants growth and development [17].…”