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Yeong Byeon Hyoung Yool Lee Kyungjin Lee Kyoungwhan Back 《Journal of pineal research》2014,57(2):219-227
Although a plant N‐acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight Arabidopsis thaliana O‐methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 mm N‐acetylserotonin (NAS). Among the eight strains harboring the full‐length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The Km and Vmax values for ASMT activity were 233 μm and 1800 pmol/min/mg protein, while the Km and Vmax values for COMT activity were 103 μm and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (Vmax/Km) for ASMT activity was 709‐fold lower than for COMT. In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose‐dependent manner, but the activity of COMT was not altered by NAS. Lastly, the Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mm NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants. 相似文献
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Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT 总被引:2,自引:0,他引:2
Yeong Byeon Hyoung Yool Lee Kyungjin Lee Sangkyu Park Kyoungwhan Back 《Journal of pineal research》2014,56(1):107-114
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. 相似文献
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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. 相似文献
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Serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin methyltransferase (ASMT) are the last two key enzymes for melatonin biosynthesis in living organisms. In this study, we demonstrated that transgenic rice (Oryza sativa L.) plants, in which expression of either endogenous SNAT or ASMT was suppressed, had reduced melatonin synthesis, confirming that both SNAT and ASMT are functionally involved in melatonin synthesis. The melatonin‐deficient SNAT rice had retarded seedling growth, which was partially restored by exogenous melatonin application, suggesting melatonin's role in seedling growth. In addition, the plants were more sensitive to various abiotic stresses, including salt and cold, compared with the wild type. Melatonin‐deficient SNAT rice had increased coleoptile growth under anoxic conditions, indicating that melatonin also inversely regulates plant growth under anaerobic conditions with the concomitant high expression of alcohol dehydrogenase genes. Similarly, the melatonin‐deficient ASMT rice exhibited accelerated senescence in detached flag leaves, as well as significantly reduced yield. These loss‐of‐function studies on the melatonin biosynthetic genes confirmed most previous pharmacological reports that melatonin not only promotes plant growth but also mitigates various abiotic stresses. 相似文献
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Chloroplast overexpression of rice caffeic acid O‐methyltransferase increases melatonin production in chloroplasts via the 5‐methoxytryptamine pathway in transgenic rice plants
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Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5‐methoxytryptamine (5‐MT) pathway. 5‐MT is known to be synthesized in vitro from serotonin by the enzymatic action of O‐methyltransferases, including N‐acetylserotonin methyltransferase (ASMT) and caffeic acid O‐methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N‐acetyltransferase (SNAT). Here, we show that 5‐MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5‐MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild‐type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5‐MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5‐MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5‐MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. 相似文献
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Abstract: Rice (Oryza sativa) N‐acetylserotonin methyltransferase (osASMT), the last enzyme in the synthesis of melatonin, was expressed in Escherichia coli and purified. We then characterized its enzyme kinetics, which is the first time this has been performed in plants. Purified glutathione S‐transferase (GST)‐fused recombinant osASMT (GST‐osASMT) and GST‐free osASMT showed specific enzyme activities of 6.6 and 12.6 pmol/min per mg protein, respectively. When evaluated by the Lineweaver‐Burk equation, GST‐free osASMT exhibited a Km of 864 μm . An in vitro enzyme assay of purified osASMT showed melatonin formation to be proportional to the enzyme and substrate concentrations, as well as time. Unlike animal ASMT, high substrate concentrations did not inhibit the activity of osASMT. Finally, melatonin biosynthesis in rice seedlings was affected by light intensity, with etiolated shoots grown in continuous darkness producing more melatonin than shoots grown in continuous light. The level of melatonin in relation to the light intensity closely paralleled the mRNA level of osASMT in the shoots, suggesting that endogenous melatonin is upregulated in darkness, as is the case in animals. 相似文献
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Yeong Byeon Dun‐Xian Tan Russel J. Reiter Kyoungwhan Back 《Journal of pineal research》2015,59(4):448-454
The cloning of the gene encoding melatonin 2‐hydroxylase (M2H), which is responsible for the synthesis of 2‐hydroxymelatonin, has expanded the study of melatonin metabolism in plants. Kinetic analysis of M2H enzymatic activity demonstrated that the catalytic efficiency of M2H is much higher than those of other melatonin biosynthetic enzymes such as serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin O‐methyltransferase (ASMT), suggesting that melatonin metabolism is rapid in plants. To test this prediction, we selected 24 plant species belonging to 16 families and quantified the levels of melatonin and 2‐hydroxymelatonin using liquid chromatography–tandem mass spectrometry (LC‐MS/MS). The melatonin levels in most of the species were <1 ng/g fresh weight (FW), while those in leaves from radish and feverfew were 3.5 and 3.3 ng/g FW, respectively. In contrast, the average levels of 2‐hydroxymelatonin were much higher at 6.2 ng/g FW. The average ratio of 2‐hydroxymelatonin to melatonin in plants was approximately 368:1, indicating that the accumulation of 2‐hydroxymelatonin predominates over that of melatonin. These data were consistent with previous results on the kinetics of the corresponding enzymes, as well as with in vivo melatonin conversion data. Among several melatonin metabolites in plants, the most abundant metabolite was found to be 2‐hydroxymelatonin (99%) followed by 4‐hydroxymelatonin (0.05%), but 6‐hydroxymelatonin was not detected in rice seedlings. 相似文献
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Sangkyu Park Yeong Byeon Hyoung Yool Lee Young‐Soon Kim Taeho Ahn Kyoungwhan Back 《Journal of pineal research》2014,57(3):348-355
Serotonin N‐acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N‐acetylserotonin, an immediate precursor for melatonin biosynthesis by N‐acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the Km (428 μm ) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis. 相似文献
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Kiyoon Kang Kyungjin Lee Sangkyu Park Young Soon Kim Kyoungwhan Back 《Journal of pineal research》2010,49(2):176-182
Abstract: Serotonin N‐acetyltransferase (SNA), a rate‐limiting enzyme in melatonin biosynthesis in vertebrates, is responsible for the production of N‐acetylserotonin; this molecule is then converted to melatonin by hydroxyindole‐O‐methyltransferase. We generated transgenic rice plants via expression of the human SNA gene under the constitutive ubiquitin promoter using Agrobacterium‐mediated gene transformation. We investigated the role of SNA in the biosynthesis of melatonin and the physiological role of melatonin in rice plants. The integration and expression of the transgene were confirmed in T1 transgenic rice seedlings by Southern, Northern, and RT‐PCR analyses. High SNA‐specific enzyme activities were observed in the transgenic rice plants, whereas the wild type revealed a trace level of SNA enzyme activity. The functional expression of SNA protein was closely associated with the elevated synthesis of N‐acetylserotonin and melatonin in the transgenic rice plants. Experiments using both exogenous treatment of serotonin and senescent detached leaves, which contain a pool of serotonin, significantly enhanced melatonin biosynthesis, indicating that endogenous serotonin levels play a bottleneck role in the pathway of melatonin biosynthesis. Finally, the transgenic rice seedlings with high levels of melatonin showed elevated chlorophyll synthesis during cold stress, suggesting a role for melatonin in cold‐stress resistance. 相似文献
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Rice histone deacetylase 10 and Arabidopsis histone deacetylase 14 genes encode N‐acetylserotonin deacetylase,which catalyzes conversion of N‐acetylserotonin into serotonin,a reverse reaction for melatonin biosynthesis in plants
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In plants, melatonin production is strictly regulated, unlike the production of its precursor, serotonin, which is highly inducible in response to stimuli, such as senescence and pathogen exposure. Exogenous serotonin treatment does not greatly induce the production of N‐acetylserotonin (NAS) and melatonin in plants, which suggests the possible existence of one or more regulatory genes in the pathway for the biosynthesis of melatonin from serotonin. In this report, we found that NAS was rapidly and abundantly converted into serotonin in rice seedlings, indicating the presence of an N‐acetylserotonin deacetylase (ASDAC). To clone the putative ASDAC gene, we screened 4 genes that were known as histone deacetylase (HDAC) genes, but encoded proteins targeted into chloroplasts or mitochondria rather than nuclei. Of 4 recombinant Escherichia coli strains expressing these genes, one E. coli strain expressing the rice HDAC10 gene was found to be capable of producing serotonin in response to treatment with NAS. The recombinant purified rice HDAC10 (OsHDAC10) protein exhibited ASDAC enzyme activity toward NAS, N‐acetyltyramine (NAT), N‐acetyltryptamine, and melatonin, with the highest ASDAC activity for NAT. In addition, its Arabidopsis ortholog, AtHDAC14, showed similar ASDAC activity to that of OsHDAC10. Both OsHDAC10 and AtHDAC14 were found to be expressed in chloroplasts. Phylogenetic analysis indicated that ASDAC homologs were present in archaea, but not in cyanobacteria, which differs from the distribution of serotonin N‐acetyltransferase (SNAT). This suggests that SNAT and ASDAC may have evolved differently from ancestral eukaryotic cells. 相似文献
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Abstract: Melatonin biosynthesis was examined in Sekiguchi mutant rice lacking functional tryptamine 5‐hydroxylase (T5H) activity, which is the terminal enzyme for serotonin biosynthesis in rice. During senescence process, the leaves of Sekiguchi mutant rice produced more tryptamine and N‐acetyltryptamine compared with the wild‐type Asahi leaves. Even though T5H activity is absent, Sekiguchi leaves produce low levels of serotonin derived from 5‐hydroxytryptophan, which was found to be synthesized during senescence process. Accordingly, both rice cultivars exhibited similar levels of N‐acetylserotonin until 6 days of senescence induction; however, only Asahi leaves continued to accumulate N‐acetylserotonin after 6 days. In contrast, a large amount of N‐acetyltryptamine was accumulated in Sekiguchi leaves, indicating that tryptamine was efficiently utilized as substrate by the rice arylalkylamine N‐acetyltransferase enzyme. An increase in N‐acetyltryptamine in Sekiguchi had an inhibitory effect on synthesis of melatonin because little melatonin was produced in Sekiguchi leaves at 6 days of senescence induction, even in the presence of equivalent levels of N‐acetylserotonin in both cultivars. The exogenous treatment of 0.1 mm N‐acetyltryptamine during senescence process completely blocked melatonin synthesis. 相似文献
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Zorica Janjetovic Zachary P. Nahmias Sherie Hanna Stuart G. Jarrett Tae‐Kang Kim Russel J. Reiter Adrzej T. Slominski 《Journal of pineal research》2014,57(1):90-102
We investigated the protective effects of melatonin and its metabolites: 6‐hydroxymelatonin (6‐OHM), N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), N‐acetylserotonin (NAS), and 5‐methoxytryptamine (5‐MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm2) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50–60%) when UVB‐exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose‐dependent increase in viability of UVB‐irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot‐blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB‐induced pyrimidine photoproducts (6‐4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser‐15 but not at Ser‐46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6‐OHM, AFMK, 5‐MT, which are endogenously produced in keratinocytes, protect these cells against UVB‐induced oxidative stress and DNA damage. 相似文献
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Gabriela S. Kinker Sueli M. Oba‐Shinjo Claudia E. Carvalho‐Sousa Sandra M. Muxel Suely K. N. Marie Regina P. Markus Pedro A. Fernandes 《Journal of pineal research》2016,60(1):84-94
Gliomas, the most common primary brain tumors in adults, are classified into four malignancy grades according to morphological features. Recent studies have shown that melatonin treatment induces cytotoxicity in glioma‐initiating cells and reduces the invasion and migration of glioma cell lines, inhibiting the nuclear factor κB (NFκB) oncopathway. Given that C6 rat glioma cells produce melatonin, we investigated the correlation between the capacity of gliomas to synthesize/metabolize melatonin and their overall malignancy. We first characterized the melatonergic system of human gliomas cell lines with different grades of aggressiveness (HOG, T98G, and U87MG) and demonstrated that glioma‐synthesized melatonin exerts an autocrine antiproliferative effect. Accordingly, the sensitivity to exogenous melatonin was higher for the most aggressive cell line, U87MG, which synthesized/accumulated less melatonin. Using The Cancer Genome Atlas RNAseq data of 351 glioma patients, we designed a predictive model of the content of melatonin in the tumor microenvironment, the ASMT:CYP1B1 index, combining the gene expression levels of melatonin synthesis and metabolism enzymes. The ASMT:CYP1B1 index negatively correlated with tumor grade, as well as with the expression of pro‐proliferation and anti‐apoptotic NFκB target genes. More importantly, the index was a grade‐ and histological type‐independent prognostic factor. Even when considering only high‐grade glioma patients, a low ASMT:CYP1B1 value, which suggests decreased melatonin and enhanced aggressiveness, was strongly associated with poor survival. Overall, our data reveal the prognostic value of the melatonergic system of gliomas and provide insights into the therapeutic role of melatonin. 相似文献