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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. [ABSTRACT FROM AUTHOR]

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate's aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity. [ABSTRACT FROM AUTHOR]

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate’s aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.

Key Message: This study provides novel insights into the evolution, diversification, and functions of melatonin biosynthesis genes in Prunus species, highlighting their potential role in regulating bud dormancy and abiotic stresses. The biosynthesis of melatonin (MEL) in plants is primarily governed by enzymatic reactions involving key enzymes such as serotonin N-acetyltransferase (SNAT), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT) and tryptophan decarboxylase (TDC). In this study, we analyzed Melatonin genes in four Prunus species such as Prunus avium (Pavi), Prunus pusilliflora (Ppus), Prunus serulata (Pser), and Prunus persica (Pper) based on comparative genomics approach. Among the four Prunus species, a total of 29 TDCs, 998 T5Hs, 16 SNATs, and 115 ASMTs within the genome of four Prunus genomes. A thorough investigation of melatonin-related genes was carried out using systematic biological methods and comparative genomics. Through phylogenetic analysis, orthologous clusters, Go enrichment, syntenic relationship, and gene duplication analysis, we discovered both similarities and variations in Melatonin genes among these Prunus species. Additionally, our study revealed the existence of unique subgroup members in the Melatonin genes of these species, which were distinct from those found in Arabidopsis genes. Furthermore, the transcriptomic expression analysis revealed the potential significance of melatonin genes in bud dormancy regulation and abiotic stresses. Our extensive results offer valuable perspectives on the evolutionary patterns, intricate expansion, and functions of PavMEL genes. Given their promising attributes, PavTDCs, PavT5H, PavNAT, and three PavASMT genes warrant in-depth exploration as prime candidates for manipulating dormancy in sweet cherry. This was done to lay the foundation for future explorations into the structural and functional aspects of these factors in Prunus species. This study offers significant insights into the functions of ASMT, SNAT, T5H, and TDC genes and sheds light on their roles in Prunus avium. Moreover, it established a robust foundation for further exploration functional characterization of melatonin genes in fruit species., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2 , are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks. [Display omitted] • Mel concentration increases in the skin of sticklebacks and flounders exposed to oxidative stress. • Mel is not synthesized in flounder's skin. • The source of increased skin Mel in sticklebacks and flounders exposed to oxidative stress is outside the skin. • Cortisol is involved in the response to oxidative stress in sticklebacks but not in flounders. [ABSTRACT FROM AUTHOR]

Melatonin ( N -acetyl-5-methoxytryptamine, MEL), its kynurenic ( N 1 -formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N 2 -formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N -phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients.

Background: Serotonin (5-HT) is an important mediator in the gastrointestinal tract, acting on different neuronal 5-HT receptors. The ionotropic 5-HT 3 receptor mediates immediate but transient spike discharge in human enteric neurons. We studied the role of the metabotropic 5-HT 1P , 5-HT 4 , and 5-HT 7 receptors to activate human submucous neurons., Methods: Neuroimaging using the voltage sensitive dye Di-8-ANEPPS was performed in submucous plexus preparations from human surgical specimens of the small and large intestine. We synthesized a new, stable 5-HT 1P agonist, 5-benzyloxyhydrazonoindalpine (5-BOHIP)., Key Results: 5-HT evoked a fast and late-onset spike discharge in enteric neurons. The fast component was blocked by the 5-HT 3 receptor antagonist cilansetron, while the remaining sustained response was significantly reduced by the 5-HT 1P receptor antagonist 5-hydroxytryptophanyl-5-hydroxytryptophan amide (5-HTP-DP). The newly synthesized 5-HT 1P agonist 5-BOHIP induced a slowly developing, long-lasting activation of submucous neurons, which was blocked by 5-HTP-DP. We could not demonstrate any 5-HT 7 receptor-induced spike discharge based on the lack of response to 5-carboxamidotryptamine. Similarly, the 5-HT 4 agonists 5-methoxytryptamine and prucalopride evoked no immediate or late-onset spike discharge., Conclusions & Inferences: Our work demonstrated for the first time the presence of functional 5-HT 1P receptors on human submucous neurons. Furthermore, we found no evidence for a role of 5-HT 4 or 5-HT 7 receptors in the postsynaptic activation of human submucous neurons by 5-HT., (© 2022 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Melatonin (N-acetyl-5-methoxytryptamine) is an indole molecule widely found in animals and plants. It is well known that melatonin improves plant resistance to various biotic and abiotic stresses due to its potent free radical scavenging ability while being able to modulate plant signaling and response pathways through mostly unknown mechanisms. In recent years, an increasing number of studies have shown that melatonin plays a crucial role in improving crop quality and yield by participating in the regulation of various aspects of plant growth and development. Here, we review the effects of melatonin on plant vegetative growth and reproductive development, and systematically summarize its molecular regulatory network. Moreover, the effective concentrations of exogenously applied melatonin in different crops or at different growth stages of the same crop are analysed. In addition, we compare endogenous phytomelatonin concentrations in various crops and different organs, and evaluate a potential function of phytomelatonin in plant circadian rhythms. The prospects of different approaches in regulating crop yield and quality through exogenous application of appropriate concentrations of melatonin, endogenous modification of phytomelatonin metabolism-related genes, and the use of nanomaterials and other technologies to improve melatonin utilization efficiency are also discussed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Research on age-dependent changes in pineal activity has been limited almost exclusively to melatonin (MLT). This study determined, for the first time, the alterations occurring in the metabolic profile of MLT synthesis-related indoles during the post-embryonic development period in birds. Turkeys reared under a 12 h light/dark cycle were euthanized at 2 h intervals for 24 h at the ages of 2, 7, 14, and 28 days and 10, 20, 30, and 45 weeks. The results showed prominent changes in the metabolic profile of indoles during development and could be distinguished in four stages. The first stage, from hatching to the age of 2 weeks, was characterized by a decrease in the 5-hydroxytryptophan concentration and an increase in the concentrations of serotonin (5-HT), MLT, 5-methoxyindoleacetic acid, and 5-methoxytryptamine (5-MTAM). During the second stage, around the age of 1 month, the concentrations of N-acetylserotonin (NAS) and MLT reached a maximum. The synthesis and degradation of 5-HT were also the highest. The third stage, around the age of 10 weeks, was characterized by decreased levels of 5-HT (approximately 50%) and 5-hydroxyindoleacetic acid and a high level of 5-MTAM. The last stage, covering the age of 20 to 45 weeks, was characterized by a large decrease in the synthesis, content, and degradation of 5-HT. Despite these changes, there were no prominent differences in the nocturnal levels of NAS and MLT between the third and fourth stages. The concentrations of all tryptophan derivatives showed daily fluctuations until the age of 45 weeks.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that possesses a wide range of biological effects. Most of the main recognized effects of this hormone in mammals are due to its interaction with two G protein-coupled receptors, MT1 and MT2. Ligand-binding studies have been based on the use of its radioligand analog, 2[125I]-iodomelatonin, a super agonist discovered in the early 1990s. This compound has been used in most of the binding studies reported in the literature. Nevertheless, more recently other possibilities arose. This chapter is a brief summary of those alternative radioligands and of their benefits one can find in using them., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Melatonin, (N-acetyl-5-methoxytryptamine), is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule. For a long time, melatonin had been described as a ligand of a putative "receptor" present in mammalian brains named MT 3 . Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor." The experimental setup of the binding measurements is unusual. The present chapter aims at describing this technique. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2)., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)