Kyoungwhan Back’s research while affiliated with Chonnam National University and other places

What is this page?


This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Publications (180)


Melatonin-induced brassinosteroid biosynthesis enhances seed size, senescence tolerance, and salt stress susceptibility in transgenic rice plants overexpressing an archaeal serotonin N-acetyltransferase
  • Article

December 2024

·

4 Reads

Melatonin Research

Kyungjin Lee

·

Kyoungwhan Back

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme catalyzing serotonin into N-acetylserotonin in the melatonin biosynthetic pathway. Recently, an archaeal SNAT from Thermoplasma volcanium (TvSNAT) was cloned and rice lines with its ectopic overexpression (TvSNAT-OE) exhibited higher melatonin levels and generated larger seeds than those of the wild type (WT). In this study, we hypothesized that the increased seed size in TvSNAT-OE rice line might be linked to enhanced brassinosteroid (BR) synthesis since melatonin level is positively associated with BR production. In rice seedlings, roots were shorter and the lamina angle was larger in TvSNAT-OE lines than those in the WT. Also, the second leaves of seedlings were longer in the TvSNAT-OE lines than that in the WT, supporting BR elevation in the TvSNAT-OE lines. In parallel with these phenotypic features, BR levels were higher in the TvSNAT-OE lines than that in the WT. Increased BR levels were associated with enhanced expression of DWARF4 and BZR1, the key genes for BR biosynthesis and signaling, in the TvSNAT-OE lines compared with the WT. Consequently, the TvSNAT-OE lines showed delayed senescence, but were more susceptible to salt stress than the WT due to enhanced BR levels.


Figure 4. SNAT enzyme kinetic analysis. Serotonin N-acetyltransferase enzyme activity as a function of (A) various temperature, (B) various pH, (C) K m and V max values for serotonin substrate, (D) K m and V max values for 5-methoxytryptamine (5-MT) substrate. Values are means ± SD (n = 3). nd, not detectable.
Figure 8. Sequence comparison and phylogenetic tree of SNAT in the Ciliophora and dinoflagellates. (A) Consensus amino acid sequences among three SNAT proteins including the human Naa50, the ciliate Stylonichia lemnae SNAT, and the dinoflagellate Polarella glacialis SNAT. Bold red letters indicate consensus amino acids. Dashes denote gaps for maximizing alignment of conserved residues. A coenzyme-A-binding pocket is underlined. (B) Phylogenetic tree analysis of SNAT proteins from the ciliates and dinoflagellates. GenBank accession numbers of various SNAT genes are as follows: human Naa50 (BAB14397), Cladocopium goreaui (CAI3999280); Effrenium voratum (CAJ1361560); Polarella glacialis (CAK0876941); Stylonichia lemna (CCKQ01002460); Paramecium sonneborni (CAD8056267); Pseudocohnilembus persalius (KRX00195); Tetrahymena thermophila SB210 (XP_001025216); Ichthyophthirius multifiliis (XP-004035125). The scale bar represents 0.3 substitutions per site.
Functional Characterization of the Ciliate Stylonychia lemnae Serotonin N-Acetyltransferase, a Pivotal Enzyme in Melatonin Biosynthesis and Its Overexpression Leads to Peroxidizing Herbicide Tolerance in Rice
  • Article
  • Full-text available

September 2024

·

6 Reads

·

1 Citation

Antioxidants

Serotonin N-acetyltransferase (SNAT) is a pivotal enzyme for melatonin biosynthesis in all living organisms. It catalyzes the conversion of serotonin to N-acetylserotonin (NAS) or 5-methoxytrypytamine (5-MT) to melatonin. In contrast to animal- and plant-specific SNAT genes, a novel clade of archaeal SNAT genes has recently been reported. In this study, we identified homologues of archaeal SNAT genes in ciliates and dinoflagellates, but no animal- or plant-specific SNAT homologues. Archaeal SNAT homologue from the ciliate Stylonychia lemnae was annotated as a putative N-acetyltransferase. To determine whether the putative S. lemnae SNAT (SlSNAT) exhibits SNAT enzyme activity, we chemically synthesized and expressed the full-length SlSNAT coding sequence (CDS) in Escherichia coli, from which the recombinant SlSNAT protein was purified by Ni2+ affinity column chromatography. The recombinant SlSNAT exhibited SNAT enzyme activity toward serotonin (Km = 776 µM) and 5-MT (Km = 246 µM) as substrates. Furthermore, SlSNAT-overexpressing (SlSNAT-OE) transgenic rice plants showed higher levels of melatonin synthesis than wild-type controls. The SlSNAT-OE rice plants exhibited delayed leaf senescence and tolerance against treatment with the reactive oxygen species (ROS)-inducing herbicide butafenacil by decreasing hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels, suggesting that melatonin alleviates ROS production in vivo.

Download

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice

May 2024

·

17 Reads

·

2 Citations

International Journal of Molecular Sciences

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.


Figure 1. (A) Amino acid sequence alignment of TvSNAT and E. coli RimI (SNAT). The conserved acetyl coenzyme A binding sites are underlined. Plus signs (+) denote similar amino acids. Dashes denote gaps. (B) Phylogenetic tree showing E. coli SNAT, archaeal orthologues and rice SNAT genes. The scale bar represents 0.3 substitutions per site. GenBank accession numbers are: TvSNAT (NC_002689), E. coli RimI (WP_137442509), rice SNAT1 (AK059369), rice SNAT2 (AK068156), human Naa50 (BAB14397), and Arabidopsis Naa50 (NM_121172).
Figure 5. Growth curves of control (pET28b) and EcRimI overexpression (pET28b-RimI) E. coli strains at (A) 28 °C, (B) 37 °C, and (C) 42 °C. Each point represents three independent replicates. E. coli was grown in nutrient-rich TB medium.
Figure 5. Growth curves of control (pET28b) and EcRimI overexpression (pET28b-RimI) E. coli strains at (A) 28 • C, (B) 37 • C, and (C) 42 • C. Each point represents three independent replicates. E. coli was grown in nutrient-rich TB medium. Biomolecules 2023, 13, x FOR PEER REVIEW 9 of 15
Figure 6. (A) Melatonin production of the control (pET28b) and EcRimI overexpression (pET28b-RimI) strains at 24 h under various culture temperature conditions. (B) Time course of melatonin production by the control (pET28b) and EcRimI overexpression (pET28b-RimI) strains at 37 • C. Medium fractions were subjected to HPLC analysis for melatonin quantification. Different letters indicate significant differences from the wild type (Tukey's honest significant difference (HSD) test; p < 0.05). Values are presented as means ± SD (n = 3).
Enzyme kinetics of SNAT proteins from various organism.
Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis

May 2023

·

46 Reads

·

4 Citations

Biomolecules

Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 μM and 528 pmol/min/mg protein toward serotonin and 201 μM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.


Figure 6. (A) Shoot length in the presence of mannitol. (B) Expression levels of genes involved in antioxidant and chaperone systems under non-stress and mannitol stress conditions. (C) Quantitative real-time polymerase chain reaction (qRT-PCR) analyses under non-stress condition. GenBank accession numbers: APX1, ascorbate peroxidase 1 (AB050724); APX4, (Os08g0549100); ABI5, abscisic
Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice

January 2023

·

27 Reads

·

4 Citations

Antioxidants

A new clade of serotonin N-acetyltransferase (SNAT), the penultimate enzyme in the melatonin biosynthetic pathway, has been reported in the archaeon Thermoplasma volcanium. The closest homolog of archaea SNAT in human was an N-alpha-acetyltransferase50 (Naa50). To determine whether human Naa50 (hNaa50) shows SNAT enzyme activity, we chemically synthesized and expressed the hNaa50 gene in Escherichia coli, followed by Ni2+ affinity purification. Purified recombinant hNaa50 showed SNAT activity (Km and Vmax values of 986 μM and 1800 pmol/min/mg protein, respectively). To assess its in vivo function, hNaa50 was overexpressed in rice (hNaa50-OE). The transgenic rice plants produced more melatonin than nontransgenic wild-type rice, indicating that hNaa50 is functionally coupled with melatonin biosynthesis. Due to its overproduction of melatonin, hNaa50-OE had a higher tolerance against osmotic stress than the wild type. Enhanced expression of the chaperone genes BIP1 and CNX in hNaa50-OE plants was responsible for the increased tolerance. It is concluded that hNaa50 harbors serotonin N-acetyltransferase enzyme activity in addition to its initial N-alpha-acetyltransferase, suggesting the bifunctionality of the hNaa50 enzyme toward serotonin and protein substrates. Consequently, ectopic overexpression of hNaa50 in rice enhanced melatonin synthesis, indicating that hNaa50 is in fact involved in melatonin biosynthesis.


Figure 1. Schematic diagram of melatonin biosynthesis. AANAT is also known as serotonin Nacetyltransferase (SNAT) in plants. ASMT, N-acetylserotonin O-methyltransferase.
Figure 1. Schematic diagram of melatonin biosynthesis. AANAT is also known as serotonin Nacetyltransferase (SNAT) in plants. ASMT, N-acetylserotonin O-methyltransferase. Antioxidants 2022, 11, x FOR PEER REVIEW 6 of 15
Figure 2. Nucleotide sequence alignments of native (black writing; AB474787) and synthetic CrAANAT (red writing) genes. Identity is denoted by red dashes. The nucleotide sequence of synthetic CrAANAT was codon-optimized with reference to rice SNAT2 (AK068156).
Figure 4. Conversion of serotonin to N-acetylserotonin as a function of (A) pH and (B) tempe (C) Substrate affinity (Km) and maximum reaction rate (Vmax) values of CrAANAT. Purified r binant GST-CrAANAT (1 μg) was incubated at a range of pH values and temperatures for 3 Km and Vmax values were determined using Lineweaver-Burk plots. N-Acetylserotonin, an i
Figure 5. Localization of CrAANAT. (A) Red fluorescence of CrAANAT-mCherry. (B) Green fluorescence of cytoplasmic GFP. (C) Cyan fluorescence of chloroplasts. (D) Merged image (A + B + C). Leaves of 30-day-old tobacco (Nicotiana benthamiana) seedlings were infiltrated with Agrobacterium (GV2260) containing XVE-inducible CrAANAT-mCherry, or constitutive 35S:GFP (cytosolic marker). Bars, 20 µm. Synthetic CrAANAT was used in place of native CrAANAT (AB474787).
Functional Characterization of Arylalkylamine N-Acetyltransferase, a Pivotal Gene in Antioxidant Melatonin Biosynthesis from Chlamydomonas reinhardtii

August 2022

·

41 Reads

·

7 Citations

Antioxidants

Arylalkylamine N-acetyltransferase (AANAT) is a pivotal enzyme in melatonin biosynthesis that catalyzes the conversion of serotonin to N-acetylserotonin. Homologs of animal AANAT genes are present in animals, but not in plants. An AANAT homolog was found in Chlamydomonas reinhardtii, but not other green algae. The characteristics of C. reinhardtii AANAT (CrAANAT) are unclear. Here, full-length CrAANAT was chemically synthesized and expressed in Escherichia coli. Recombinant CrAANAT exhibited AANAT activity with a Km of 247 μM and Vmax of 325 pmol/min/mg protein with serotonin as the substrate. CrAANAT was localized to the cytoplasm in tobacco leaf cells. Transgenic rice plants overexpressing CrAANAT (CrAANAT-OE) exhibited increased melatonin production. CrAANAT-OE plants showed a longer seed length and larger second leaf angle than wild-type plants, indicative of the involvement of brassinosteroids (BRs). As expected, BR biosynthesis- and signaling-related genes such as D2, DWARF4, DWARF11, and BZR1 were upregulated in CrAANAT-OE plants. Therefore, an increased endogenous melatonin level by ectopic overexpression of CrAANAT seems to be closely associated with BR biosynthesis, thereby influencing seed size.


Figure 1. Reaction catalyzed by M3H and amino acid sequences of rice OsM3H and Arabidop AtM3H. (A) Enzymatic conversion of M2H and M3H. (B) Amino acid sequences of OsM3H a AtM3H. Asterisks, identical amino acids; dashes, gaps; underlining, conserved 2-ODD doma GenBank accession numbers of OsM3H and AtM3H, AK067086 and AT1g17020, respectively.
The Antioxidant Cyclic 3-Hydroxymelatonin Promotes the Growth and Flowering of Arabidopsis thaliana

June 2022

·

40 Reads

·

11 Citations

Antioxidants

In plants, melatonin is metabolized into several compounds, including the potent antioxidant cyclic 3-hydroxymelatonin (3-OHM). Melatonin 3-hydroxylase (M3H), a member of the 2-oxo-glutarate-dependent enzyme family, is responsible for 3-OHM biosynthesis. Although rice M3H has been cloned, its roles are unclear, and no homologs in other plant species have been characterized. Here, we cloned and characterized Arabidopsis thaliana M3H (AtM3H). The purified recombinant AtM3H exhibited Km and Vmax values of 100 μM and 20.7 nmol/min/mg protein, respectively. M3H was localized to the cytoplasm, and its expression peaked at night. Based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, 3-OHM exhibited 15-fold higher antioxidant activity than melatonin. An Arabidopsis M3H knockout mutant (m3h) produced less 3-OHM than the wildtype (WT), thus reducing antioxidant activity and biomass and delaying flowering. These defects were caused by reduced expression of FLOWERING LOCUS T (FT) and gibberellin-related genes, which are responsible for flowering and growth. Exogenous 3-OHM, but not exogenous melatonin, induced FT expression. The peak of M3H expression at night matched the FT expression pattern. The WT and m3h exhibited similar responses to salt stress and pathogens. Collectively, our findings indicate that 3-OHM promotes growth and flowering in Arabidopsis.


Figure 1. Effects of various hormones on melatonin synthesis. (A) Effects of BR on melatonin content. Brassinazole (BZ), a BR biosynthesis inhibitor, was used at a concentration of 100 μM. (B) Effects of ethylene on melatonin content. (C) Effects of 6-benzylaminopurine (BA) on melatonin content. (D) Effects of indole-3-acetic acid (IAA) on melatonin content. Seven-day-old rice seedlings were treated rhizosperically with varying levels of hormones independently for 24 h and transferred to new 50 mL conical tubes containing 0.5 mM cadmium for 3 days to induce melatonin synthesis. Different letters denote significant differences (p < 0.05) as determined by Tukey's HSD post hoc test. C, water containing 0.1% ethanol. Ethephon that releases ethylene was used for ethylene treatment.
Figure 2. Cont.
Figure 8. Effects of exogenous BR and GA in RAVL1 and Gα RNAi transgenic rice seedlings. (A-C Phenotypes of 7-day-old rice seedlings after cadmium treatment in response to exogenous BR and GA3 treatments. (D) Melatonin levels of control-, BR-, and GA-treated rice seedlings. Seven-day-old wild-type (WT) and RNAi rice seedlings were challenged with either BR or GA for 24 h. The result ing rice seedlings were treated with 0.5 mM cadmium for 3 days under conditions of continuous light at 28 °C followed by HPLC quantification of melatonin. Different letters denote significan differences (p < 0.05), as determined by Tukey's HSD post hoc tests. Mock, water containing 0.1% ethanol; BR, 1 μM epibrassinolide; GA3, 10 μM gibberellic acid 3.
Figure 9. Proposed model for melatonin biosynthesis by endogenous hormones. (A) Brassinosteroids (BR)-induced melatonin increase in a gibberellins (GA)-independent manner and (B) BR-induced GA-dependent regulation of growth and leaf angle. GA and BR induce melatonin production independently, whereas abscisic acid inhibits melatonin production. The induction patterns of genes responsible for melatonin biosynthesis and metabolism were slightly different between hormone treatments. BR treatment induced several genes involved in melatonin biosynthesis, including TDC1, TDC3, T5H, and ASMT1, whereas GA induced TDC3, T5H, and ASMT1. ASDAC and M2H were downregulated by GA treatment, while only ASDAC was downregulated by BR treatment. Unlike BR and GA, abscisic acid inhibits melatonin synthesis [20].
Molecular Regulation of Antioxidant Melatonin Biosynthesis by Brassinosteroid Acting as an Endogenous Elicitor of Melatonin Induction in Rice Seedlings

May 2022

·

59 Reads

·

12 Citations

Antioxidants

Gibberellic acid (GA) was recently shown to induce melatonin synthesis in rice. Here, we examined whether brassinosteroids (BRs) also induce melatonin synthesis because BRs and GA show redundancy in many functions. Among several plant hormones, exogenous BR treatment induced melatonin synthesis by twofold compared to control treatment, whereas ethylene, 6-benzylaminopurine (BA), and indole-3-acetic acid (IAA) showed negligible effects on melatonin synthesis. Correspondingly, BR treatment also induced a number of melatonin biosynthetic genes in conjunction with the suppression of melatonin catabolic gene expression. Several transgenic rice plants with downregulated BR biosynthesis-related genes, such as DWARF4, DWARF11, and RAV-Like1 (RAVL1), were generated and exhibited decreased melatonin synthesis, indicating that BRs act as endogenous elicitors of melatonin synthesis. Notably, treatment with either GA or BR fully restored melatonin synthesis in the presence of paclobutrazol, a GA biosynthesis inhibitor. Moreover, exogenous BR treatment partially restored melatonin synthesis in both RAVL1 and Gα RNAi transgenic rice plants, whereas GA treatment fully restored melatonin synthesis comparable to wild type in RAVL1 RNAi plants. Taken together, our results highlight a role of BR as an endogenous elicitor of melatonin synthesis in a GA-independent manner in rice plants.


Figure 7. Gain-of-function analysis of m2h knockout mutant seed germination following exogenous 2-hydroxymelatonin (2-OHM) treatment. Non-dormant A. thaliana WT and m2h knockout mutant seeds were germinated with 2-OHM (20 μM) or without 2-OHM (Control) at 25 °C for 40 h. (A) Photographs of germinated seeds upon various chemical treatments at 40 h after imbibition. (B) Quantification of germination in (A). Error bars indicate standard deviation (SD) of three biological replicates. Different letters indicate significant differences (p < 0.05; ANOVA, followed by Tukey's HSD post hoc tests).
2-Hydroxymelatonin Promotes Seed Germination by Increasing Reactive Oxygen Species Production and Gibberellin Synthesis in Arabidopsis thaliana

April 2022

·

79 Reads

·

18 Citations

Antioxidants

It was recently reported that 2-hydroxymelatonin (2-OHM) is responsible for inducing reactive oxygen species (ROS) in plants. ROS are crucial molecules that promote germination through interaction with hormones such as gibberellic acid (GA). In this study, to confirm the pro-oxidant role of 2-OHM, we investigated its effect on seed germination in Arabidopsis thaliana (L.) Heynh. Columbia-0. We found that 2-OHM treatment stimulated seed germination by 90% and 330% in non-dormant and dormant seeds, respectively, whereas melatonin marginally increased germination (~13%) in both seed types compared to untreated control seeds. The germination promotion effects of exogenous 2-OHM treatment were due to increased ROS production followed by the induction of GA synthesis and expression of responsive genes. Accordingly, melatonin 2-hydroxylase (M2H), the gene responsible for 2-OHM synthesis, was strictly expressed only during the germination process. Further molecular genetic analyses using m2h knockout mutant and M2H overexpression clearly supported an increase in ROS triggered by 2-OHM, followed by increased expression of GA-related genes, which shortened the time to germination. Notably, 2-OHM application to m2h knockout mutant seeds fully recovered germination to levels comparable to that of the wild type, whereas melatonin treatment failed to increase germination. Together, these results indicate that 2-OHM is a pivotal molecule that triggers increased ROS production during seed germination, thereby enhancing germination via the GA pathway in Arabidopsis thaliana.


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 (Km) and maximum reaction rate (Vmax) values of TvSNAT (also called TvArd1). TvSNAT (1 μg) was incubated at a range of pH values and temperatures for 30 min. Km and Vmax values were determined using LineweaverBurk plots. In vitro enzymatic N-acetylserotonin products were measured by high-performance liquid chromatography. 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 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).
Functional Characterization of Serotonin N-Acetyltransferase in Archaeon Thermoplasma volcanium

March 2022

·

46 Reads

·

19 Citations

Antioxidants

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.


Citations (97)


... Melatonin synthesis ( Figure 2) is a sequential process, consisting of four phases/steps [37], and begins in the first phase with the transformation of tryptophan into 5-hydroxytryptophan, a transformation that occurs due to the action of the enzyme tryptophan hydroxylase (TPH). Subsequently, 5-hydroxytryptophan is transformed into serotonin (5-HT), and after transformation, serotonin undergoes the process of N-acetylation as a result of the action of arylalkylamine N-acetyltransformase (AANAT), forming NAS (N-acetylserotonin). ...

Reference:

Melatonin: An Overview on the Synthesis Processes and on Its Multiple Bioactive Roles Played in Animals and Humans
Functional Characterization of the Ciliate Stylonychia lemnae Serotonin N-Acetyltransferase, a Pivotal Enzyme in Melatonin Biosynthesis and Its Overexpression Leads to Peroxidizing Herbicide Tolerance in Rice

Antioxidants

... In recent years, microbial heterologous synthesis of melatonin has emerged as a research hotspot due to its cost-effectiveness and sustainability. Additionally, biotechnological advancements in rice have demonstrated significant potential to enhance the efficiency of melatonin synthesis [51]. This approach offers a novel strategy for the development of vaccines against the novel coronavirus. ...

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice

International Journal of Molecular Sciences

... Later experiments showed that RimI is able to modify the elongation factor Tu in E. coli, and hence is involved in the regulation of the protein synthesis in this organism (Pletnev et al., 2022). Knock down mutation of rimI in E. coli does not affect growth, while overexpression enhances growth (Lee and Back, 2023). The RimI protein from Mycobacterium tuberculosis displays a relaxed substrate specificity, i.e. it is able to acetylate proteins other than its original target, the S18 ribosomal protein (Pathak et al., 2016), and thus is candidate to regulate bacterial processes other than those first described for this enzyme. ...

Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis

Biomolecules

... As for pET300-SlSNAT vector construction, the full-length SlSNAT DNA of 546 nucleotides in length was amplified by PCR using a primer set (forward primer 5 ′ -AAA AAG CAG GCT CCA TGC CGG CGC CCG AGG-3 ′ ; reverse primer 5 ′ -AGA AAG CTG GGT CTA CTG CGA CGT GGT CGA-3 ′ ) using the synthetic SlSNAT DNA as the template. The resulting PCR product was further amplified by PCR using an attB primer set [18]. The full-length SlSNAT PCR product was cloned into the pDONR221 gateway vector (Invitrogen, Carlsbad, CA, USA) via the BP recombination reaction. ...

Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice

Antioxidants

... In the last step, N-acetylserotonin is transformed into melatonin, and this process of transformation of N-acetylserotonin into melatonin is facilitated by the enzyme hydroxyindole-O-methyltransferase (HIOMT) [38]. Due to the fact that the enzyme arylalkylamine N-acetyltransformase (AANAT), which plays the role of catalyzing the conversion of serotonin into N-acetylserotonin, has minimal activity in the organism during the day, the melatonin production process is limited, thus favoring the accumulation of serotonin in pinealocytes, while melatonin synthesis at night is due to the increased activity of the AANAT as a result of the onset of darkness [39]. ...

Functional Characterization of Arylalkylamine N-Acetyltransferase, a Pivotal Gene in Antioxidant Melatonin Biosynthesis from Chlamydomonas reinhardtii

Antioxidants

... The hydroxymetabolite 3-OHM, formed from melatonin as a result of M3H activity, has intrinsic antioxidant activity targeting hydroxyl and hydroperoxyl ( • OOH) radicals (Tan et al., 2014). In reaction with 1,1-diphenyl-2-picrylhydrazyl, 3-OHM was shown to have 15 times more antioxidant activity than melatonin (Lee and Back, 2022). ...

The Antioxidant Cyclic 3-Hydroxymelatonin Promotes the Growth and Flowering of Arabidopsis thaliana

Antioxidants

... Our study further explored how exogenous application of MT influences the endogenous levels of BRs and how exogenous BL affects the endogenous content of MT, revealing a positive correlation between these phytohormones. These findings align with the work of Hwang and Back [75], who found that exogenous BL induced the biosynthesis of MT in rice seedlings under both Cd stress and non-stress conditions. Additionally, exogenous MT was shown to induce the expression of several BR biosynthetic genes, while the content of bioactive BRs was reduced in melatonin-deficient transgenic rice plants [76]. ...

Molecular Regulation of Antioxidant Melatonin Biosynthesis by Brassinosteroid Acting as an Endogenous Elicitor of Melatonin Induction in Rice Seedlings

Antioxidants

... These results confirm that ABI4 is an important factor in the regulation of the interactions between melatonin and ABA. However, the exact biological meaning of ABA-dependent shifts in the expression of melatonin catabolism genes remains to be clarified, taking into account that, on the one hand, 2-OHM triggers the production of ROS [35], and, on the other hand, that 3-OHM is a powerful antioxidant exhibiting even higher antioxidant activity than melatonin [5]. ...

2-Hydroxymelatonin Promotes Seed Germination by Increasing Reactive Oxygen Species Production and Gibberellin Synthesis in Arabidopsis thaliana

Antioxidants

... The purified recombinant Trx-SlSNAT protein was first examined for SNAT enzyme activity in catalyzing the conversion of serotonin to NAS. As shown in Figure 3B, the recombinant SlSNAT exhibited SNAT-specific enzyme activity of 7.1 pkat/mg protein, which was similar to the activity reported previously for an archaeon SNAT (6.7 pkat/mg protein) [20]. The SNAT enzyme activity of SlSNAT was 1.8-fold higher than that of E. coli RimI [21] but 4.7-fold lower than that of rice SNAT3 [28]. ...

Functional Characterization of Serotonin N-Acetyltransferase in Archaeon Thermoplasma volcanium

Antioxidants

... BIRC3, or Baculoviral IAP Repeat Containing 3, belongs to the inhibitor of apoptosis protein family, pivotal in apoptosis regulation, cell survival promotion, and intracellular signaling maintenance 38 . Dysregulated BIRC3 expression significantly correlates with tumorigenesis, progression, and treatment resistance, notably through NF-κB pathway regulation, influencing inflammatory and immune responses crucial in tumor progression 39 . ...

Phytomelatonin as a signaling molecule for protein quality control via chaperone, autophagy, and ubiquitin–proteasome systems in plants
  • Citing Article
  • March 2022

Journal of Experimental Botany