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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
Source publication
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...
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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...
Citations
... 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. ...
Introduction
Cutibacterium acnes can both be a helpful colonizer of the human skin as well as the causative agent of acne and purulent infections. Until today, it is a moot point whether there are C. acnes strains exclusively devoted to be part of the skin microbiome and others, that carry special features enabling them to cause disease. So far, the search for the molecular background of such diverse behavior has led to inconsistent results.
Methods
In the present study, we prospectively collected C. acnes strains from 27 infected persons and 18 healthy controls employing rigid selection criteria to ensure their role as infectious agent or colonizer. The genome sequences from these strains were obtained and carefully controlled for quality.
Results
Deduced traditional phylotyping assigned almost all superficial isolates to type IA1, while the clinical strains were evenly distributed between types IA1, IB, and II. Single locus sequence typing (SLST) showed a predominance of A1 type for the control strains, whereas 56% of the clinical isolates belonged to types A1, H1 and K8. Pangenome analysis from all the present strains and 30 published genomes indicated the presence of an open pangenome. Except for three isolates, the colonizing strains clustered in clades separate from the majority of clinical strains, while 4 clinical strains clustered with the control strains. Identical results were obtained by a single nucleotide polymorphism (SNP) analysis. However, there were no significant differences in virulence gene contents in both groups.
Discussion
Genome-wide association studies (GWAS) from both the pangenome and SNP data consistently showed genomic differences between both groups located in metabolic pathway and DNA repair genes. Thus, the different behavior of colonizing and infectious C. acnes strains could be due to special metabolic capacities or flexibilities rather than specific virulence traits
... Both the pET32b-SlSNAT and pET300-SlSNAT plasmids were transformed into E. coli strain BL21(DE3) (Invitrogen, Carlsbad, CA, USA). Further E. coli culture, isopropylβ-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO, USA) treatment, and affinity (Ni 2+ ) purification were described in detail previously [21]. The purified thioredoxin (Trx)tagged SlSNAT fusion protein was mixed with the equal volume of glycerol and stored at −80 • C until use. ...
... The putative SlSNAT was annotated as a member of the protein NAT family carrying a region with identity to E. coli RimI, ranging in size from 40 to 176 aa. It was recently reported that RimI, an N-terminal protein acetyltransferase, also exhibited SNAT enzyme activity [21]. Taken together, the results of these in silico analyses suggested that the putative SlSNAT may exhibit SNAT enzyme activity. ...
... The putative SlSNAT was annotated as a member of the protein NAT family carrying a region with identity to E. coli RimI, ranging in size from 40 to 176 aa. It was recently reported that RimI, an Nterminal protein acetyltransferase, also exhibited SNAT enzyme activity [21]. Taken together, the results of these in silico analyses suggested that the putative SlSNAT may exhibit SNAT enzyme activity. ...
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.
... Melatonin is found in almost all living organisms, including animals, bacteria, archaea, and plants [19][20][21][22]. In animals, melatonin acts as a neurohormone, influencing circadian rhythms and seasonal reproduction [23]; other functions include energy metabolism, as well as anti-inflammatory, anti-cancer, and anti-aging effects [24]. ...
... SNAT2, but not SNAT1, is associated with brassinosteroid synthesis [57]. Importantly, SNAT1 and SNAT2 are specific to plants, whereas SNAT3 orthologs are universally present in a diverse array of organisms, including rice (this report), archaea [21], humans [35], and Escherichia coli [22] (Figure 8). Our study identified rice SNAT3 as a functional ortholog of archaeal SNAT. ...
... As for the pET300 vector, full-length OsSNAT3 cDNA was amplified by PCR by using a primer set (OsSNAT3 forward primer, 5′-AAA AAG CAG GCT CCA TGG GCG CCG GGG AAG-3′; OsSNAT3 reverse primer, 5′-AGA AAG CTG GGT TCA TTT CTT TGT AGC-3′) with a template plasmid containing OsSNAT3 cDNA provided by the National Institute of Agrobiological Sciences. The first PCR product was used for the template of the second PCR using the attB primer set, as described previously [22]. The second OsSNAT3 PCR product was cloned using gateway recombination reactions in the pDONR221 vector (Invitrogen, Carlsbad, CA, USA) to generate pDONR221-OsSNAT3 plasmid, and then recombined into the destination vector pET300/NT-DEST (Invitrogen) resulting in the pET300-OsSNAT3 plasmid, according to the manufacturer's procedure. ...
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.
... This discovery, coupled with the observations of the presence of a major enzyme required for melatonin synthesis in bacteria [16,17] the evolutionary precursors of eukaryotic mitochondria [18] led to the prediction that these organelles in every eukaryotic cell are the likely site of the intracellular synthesis of melatonin [19]. The theory related to the mitochondrial site of melatonin synthesis has been further bolstered by recent observations documenting that bacteria not only have the enzymes for melatonin generation but actually produce the molecule [20,21]. ...
