Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT

Abstract

Serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) are the final two enzymes in the melatonin synthesis pathway in plants. Although their corresponding genes have been cloned, their cellular localization and enzymatic characteristics are unknown. Using confocal microscopy, we showed that SNAT protein is localized in chloroplasts, whereas ASMT is expressed in the cytoplasm. In vitro measurement of ASMT enzyme activity revealed a peak of activity in roots, but SNAT enzyme activity was not detected in any plant tissues. This may be attributed in part to an effect of chlorophyll because SNAT enzyme activity was greatly inhibited by chlorophyll in a dose-dependent manner. Because the SNAT protein of cyanobacteria is thermophilic, we examined the effect of temperature on the activity of the rice SNAT and ASMT enzymes. Purified recombinant rice SNAT and ASMT enzymes had an optimum temperature for activity of 55°C. The Km and Vmax values for SNAT at 55°C were 270 μm and 3.3 nmol/min/mg protein, whereas the Km and Vmax for ASMT were 222 μm and 9 nmol/min/mg protein, respectively. The catalytic efficiency (Vmax /Km ) values of SNAT and ASMT were 16-fold and 4054-fold higher at 55°C than at 30°C suggestive of increased melatonin production at high temperature in plants.

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.…”

“…Both plasmids were transformed into the Agrobacterium tumefaciens strain GV2260 using the freeze-thaw method, and transient expression analyses were performed as described by Voinnet et al [28]. Two-week-old tobacco (Nicotiana benthamiana) leaves were infiltrated with Agrobacterium strains followed by infiltration with 10 lM b-estradiol for transgene induction, as described previously [3]. Images were generated using a Leica TCS-SP5 confocal microscope (Leica Microsystems, Exton, PA, USA).…”

“…The pER-mCherry vector (a kind gift from Dr. H.G. Kang, Texas State University, San Marcos, TX, USA) was used for subcellular localization analysis, as described previously [3]. Briefly, full-length AtSNAT cDNA was PCR amplified using the primer set containing an AscI site (forward 5ʹ-GGG GGC GCG CCA TGC TAC TAA TCC CAA-3ʹ; reverse 5ʹ-GGC GCG CCG CTT TGG GTA CCA AAA-3ʹ).…”

“…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].…”