Functional Characterization of Serotonin N-Acetyltransferase in Archaeon Thermoplasma volcanium

Abstract

Serotonin N-acetyltransferase is the penultimate enzyme in the melatonin biosynthetic pathway that catalyzes serotonin into N-acetylserotonin. Many SNAT genes have been cloned and characterized from organisms ranging from bacteria to plants and mammals. However, to date, no SNAT gene has been identified from Archaea. In this study, three archaeal SNAT candidate genes were synthesized and expressed in Escherichia coli, and SNAT enzyme activity was measured using their purified recombinant proteins. Two SNAT candidate genes, from Methanoregulaceae (Archaea) and Pyrococcus furiosus, showed no SNAT enzyme activity, whereas a SNAT candidate gene from Thermoplasma volcanium previously named TvArd1 exhibited SNAT enzyme activity. The substrate affinity and the maximum reaction rate of TvSNAT toward serotonin were 621 μM and 416 pmol/min/mg protein, respectively. The highest amine substrate was tyramine, followed by tryptamine, serotonin, and 5-methoxytryptamine, which were similar to those of plant SNAT enzymes. Homologs of TvSNAT were found in many Archaea families. Ectopic overexpression of TvSNAT in rice resulted in increased melatonin content, antioxidant activity, and seed size in conjunction with the enhanced expression of seed size-related gene. This study is the first to report the discovery of SNAT gene in Archaea. Future research avenues include the cloning of TvSNAT orthologs in different phyla, and identification of their regulation and functions related to melatonin biosynthesis in living organisms.

Keywords: Ard1; N-acetylserotonin; N-acetyltyramine; archaea; melatonin; rice seed size; synthetic genes.

Figure 1

Summary of synthetic archaeal GNAT genes and recombinant GNAT protein purification. (A) Modification of archaeal GNAT genes. (B) Purification of N-terminal His × 6-tagged GNAT proteins. (C) SNAT activity measurements. Bacterial host strain BL21 (DE3) cells harboring the pET30-GNAT plasmids were incubated with isopropyl β-d-1-thiogalactopyranoside (IPTG) for 5 h at 28 °C. M, molecular mass standards; lane 1, total proteins in 15-µL aliquots of bacterial culture without IPTG; lane 2, total proteins in 15-µL aliquots of bacterial culture with IPTG; lane 3, 20 µg soluble protein; lane 4, 5 µg protein purified by affinity chromatography. Protein samples were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and stained with Coomassie blue. SNAT enzyme activity was measured according to N-acetylserotonin production in the presence of 0.5 mM serotonin at 45 °C and pH 8.8.

Figure 2

SNAT enzyme activity catalyzing the conversion of serotonin to N-acetylserotonin as a function of (A) pH and (B) temperature. (C) Determination of substrate affinity (K_m) and maximum reaction rate (V_max) values of TvSNAT (also called TvArd1). TvSNAT (1 µg) was incubated at a range of pH values and temperatures for 30 min. K_m and V_max values were determined using Lineweaver–Burk plots. In vitro enzymatic N-acetylserotonin products were measured by high-performance liquid chromatography. Data are the means ± standard deviation (n = 3).

Figure 3

Schematic diagram of the SNAT reaction and substrate preference. (A) Enzymatic reaction of SNAT in two different melatonin biosynthetic pathways. (B) TvSNAT enzyme activity measurements for various substrates. SNAT enzyme activity was measured in the presence of 0.5 mM of each substrate at 45 °C and pH 8.8. Data are the means ± standard deviation (n = 3). ASMT, N-acetylserotonin O-methyltransferase; SNAT, serotonin N-acetyltransferase.

Figure 4

Effects of various substrates on SNAT activity. (A) SNAT enzyme activity of recombinant purified TvSNAT in the presence of serotonin (0.5 mM) and various amines (0.5 mM). Different letters indicate significant differences [p < 0.05; analysis of variance (ANOVA), followed by Tukey’s honest significant difference (HSD) post hoc tests]. The green, blue and red colors emphasize compounds which alter SNAT activity. (B) Dose-dependent inhibition of SNAT enzyme activity by spermidine or octopamine. SNAT activity was assayed in the presence of polyamines at various concentrations and expressed as a percentage relative to that in the absence of polyamines (Figure 4B). Data are the means ± standard deviation (n = 3).

Figure 5

Phylogenetic analysis of TvSNAT constructed using the neighbor-joining method for (A) Archaea and (B) Animalia and Plantae. Scale bars in (A,B) represent 0.3 and 0.6 substitutions per site, respectively. Numbers in parentheses are GenBank accession numbers of corresponding genes. Alignments and the phylogenetic analyses were performed using the BLAST-Explorer tool (www.phylogeny.fr, accessed on 6 November 2019)).

Figure 6

Schematic diagram of overexpression binary vectors, reverse-transcription polymerase chain reaction (RT-PCR) analyses, and melatonin content in transgenic rice. (A) Binary vector (GenBank accession no. EU161568) used for TvSNAT overexpression. (B) RT-PCR analyses of 7-day-old transgenic and wild-type (WT) rice seedlings. (C) Phenotypes of 7-day-old seedlings. (D) Melatonin content in 7-day-old seedlings. (E) Radical scavenging activity assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) in seeds. TvSNAT, Thermoplasma volcanium serotonin N-acetyltransferase; Ubi-P, maize ubiquitin promoter; HPT, hygromycin phosphotransferase; UBQ5, rice ubiquitin 5 gene (GenBank accession no. Os03g13170). Numbers in parentheses indicate the numbers of PCR cycles performed. Asterisks indicate significant differences from the WT (Tukey’s honest significant difference test; p < 0.05). RSA, radical scavenging activity.

Figure 7

Rice grain morphology and expression levels of seed size-related genes. (A) Grain lengths, (B) grain widths, and (C) 1000-grain weights of the wild-type (WT) and TvSNAT-OE lines. (D) Expression levels of rice seed size-related genes determined by reverse-transcription polymerase chain reaction (RT-PCR) in 7-day-old seedlings. (E) Seed melatonin content levels. GenBank accession numbers for GIF1, GW2, and UBQ5 are Os04g33740, Os02g14720, and Os03g13170, respectively. Numbers in parentheses indicate the numbers of PCR cycles performed. Asterisks indicate significant differences from the WT (Tukey’s honest significant difference test; p < 0.05).