Molecular cloning of rice serotonin N‐acetyltransferase, the penultimate gene in plant melatonin biosynthesis

Abstract

Because of the absence of an arylalkylamine N-acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5-related N-acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N-acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N-acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N-acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N-acetylserotonin. The values for Km and Vmax were 385 μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N-acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N-acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate-limiting enzyme of melatonin biosynthesis in plants.

Determining diurnal and seasonal changes in melatonin andtryptophan contents of eggplant (Solanum melongena L.)

“…In plants, the conversion of tryptophan to tryptamine catalyzed by the enzyme tryptophan decarboxylase is known to be the rate-limiting step (Zhao et al, 2013). On the other hand, Kang et al (2013) presented new evidence that the last step in the pathway (conversion of N-acetylserotonin to melatonin), regulated by the enzyme N-acetyl serotonin methyltransferase, determines melatonin production in plants. Phytomelatonin levels vary not only among plant species but also within varieties of the same species (Paredes et al, 2009;Posmyk and Janas, 2009;Arnao and Hernández-Ruiz, 2015b).…”

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

“…SNAT enzyme activity was measured using purified recombinant AtSNAT protein by incubating in a total volume of 100 lL containing 0.5 mM serotonin (or 5-methoxytryptamine) and 0.5 mM acetyl-CoA in 100 mM potassium phosphate (pH 8.8), as described previously [1,3,5]. Briefly, SNAT enzyme activity was measured at 45°C (or varying temperatures) for 1 hr and stopped by addition of 50 lL methanol.…”

“…Briefly, SNAT enzyme activity was measured at 45°C (or varying temperatures) for 1 hr and stopped by addition of 50 lL methanol. A 10 lL aliquot was subjected to high-performance liquid chromatography (HPLC) using the fluorescence detector system, as described previously [1,3,5]. Nonenzymatic reaction products were subtracted.…”

“…SNAT enzymes catalyze serotonin into N-acetylserotonin in chloroplasts, whereas ASMT is responsible for melatonin production from N-acetylserotonin, which occurs in the cytoplasm [3]. In addition to their different localizations, they differ in gene copy numbers in that SNAT is expressed as a single copy and ASMT is encoded by a small gene family [1,4].…”

“…Serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) are the penultimate and last enzyme in melatonin biosynthesis in plants, respectively [1,2]. SNAT enzymes catalyze serotonin into N-acetylserotonin in chloroplasts, whereas ASMT is responsible for melatonin production from N-acetylserotonin, which occurs in the cytoplasm [3].…”