Reannotation of Fly Amanita l-DOPA Dioxygenase Gene Enables Its Cloning and Heterologous Expression.

The l-DOPA dioxygenase of Amanita muscaria (AmDODA) participates in the biosynthesis of betalain- and hygroaurin-type natural pigments. AmDODA is encoded by the dodA gene, whose DNA sequence was inferred from cDNA and gDNA libraries almost 30 years ago. However, reports on its heterologous expression rely on either the original 5'-truncated cDNA plasmid or artificial gene synthesis. We provide unequivocal evidence that the heterologous expression of AmDODA from A. muscaria specimens is not possible by using the coding sequence previously inferred for dodA. Here, we rectify and reannotate the full-length coding sequence for AmDODA and express a 205-aa His-tagged active enzyme, which was used to produce the l-DOPA hygroaurin, a rare fungal pigment. Moreover, AmDODA and other isozymes from bacteria were submitted to de novo folding using deep learning algorithms, and their putative active sites were inferred and compared. The wide catalytic pocket of AmDODA and the presence of the His-His-His and His-His-Asp motifs can provide insight into the dual cleavage of l-DOPA at positions 2,3 and 4,5 as per the mechanism proposed for nonheme dioxygenases.

Biosynthesis of Guanitoxin Enables Global Environmental Detection in Freshwater Cyanobacteria.

Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.

Fluorescent patterning of paper through laser engraving.

While thermal treatment of paper can lead to the formation of aromatic structures via hydrothermal treatment (low temperature) or pyrolysis (high temperature), neither of these approaches allow patterning the substrates. Somewhere in between these two extremes, a handful of research groups have used CO2 lasers to pattern paper and induce carbonization. However, none of the previously reported papers have focused on the possibility to form fluorescent derivatives via laser-thermal engraving. Exploring this possibility, this article describes the possibility of using a CO2 laser engraver to selectively treat paper, resulting in the formation of fluorescent compounds, similar to those present on the surface of carbon dots. To determine the most relevant variables controlling this process, 3 MM chromatography paper was treated using a standard 30 W CO2 laser engraver. Under selected experimental conditions, a blue fluorescent pattern was observed when the substrate was irradiated with UV light (365 nm). The effect of various experimental conditions (engraving speed, engraving power, and number of engraving steps) was investigated to maximize the fluorescence intensity. Through a comprehensive characterization effort, it was determined that 5-(hydroxymethyl)furfural and a handful of related compounds were formed (varying in amount) under all selected experimental conditions. To illustrate the potential advantages of this strategy, that could complement those applications traditionally developed from carbon dots (sensors, currency marking, etc.), a redox-based optical sensor for sodium hypochlorite was developed.

Characterizing the Bioluminescence of the Humboldt Squid, Dosidicus gigas (d'Orbigny, 1835): One of the Largest Luminescent Animals in the World.

Bioluminescence is found in a number of cephalopods, such as Watasenia scintillans and Sthenoteuthis oualaniensis; however, many species remain poorly studied, including the Humboldt squid, Dosidicus gigas. This is the largest member of the Ommastrephidae family and grows to 2 m in length, making it one of the largest luminescent animals ever observed. Humboldt squid have small photophores all over their body that emit a brilliant blue luminescence. Using lyophilized photophores from squid caught off the coast of Chile, experiments were conducted to isolate the luciferin and protein involved in its bioluminescence. Methanolic extracts of the photophores were shown to contain dehydrocoelenterazine, and a membrane-bound photoprotein was shown to be involved. This photoprotein was purified using ion exchange chromatography, and SDS-PAGE showed a clean band of approximately 60 kDa. The excised band was analyzed by LC/MS, and the obtained data were compared against the transcriptome data of D. gigas, allowing us to find two gene products which displayed high coverage (>80%), the enzymes symplectin and vanin-2, which potentially associate with light emission process in this organism. Finally, the purified photoprotein was shown to emit a blue light (470 nm) in the presence of dehydrocoelenterazine.

Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella.

Microcystins (MCs) are serine/threonine phosphatase inhibitors synthesized by several members of the phylum Cyanobacteria. Mining the draft genome sequence of the nostocalean MC-producing Fischerella sp. strain CENA161 led to the identification of three contigs containing mcy genes. Subsequent PCR and Sanger sequencing allowed the assembling of its complete biosynthetic mcy gene cluster with 55,016 bases in length. The cluster encoding ten genes (mcyA-J) with a central bidirectional promoter was organized in a similar manner as found in other genera of nostocalean cyanobacteria. However, the nucleotide sequence of the mcy gene cluster of Fischerella sp. CENA161 showed significant differences from all the other MC-producing cyanobacterial genera, sharing only 85.2 to 74.1% identities. Potential MC variants produced by Fischerella sp. CENA161 were predicted by the analysis of the adenylation domain binding pockets and further investigated by LC-MS/MS analysis. To our knowledge, this study presents the first complete mcy cluster characterization from a strain of the genus Fischerella, providing new insight into the distribution and evolution of MCs in the phylum Cyanobacteria.

Identification of hispidin as a bioluminescent active compound and its recycling biosynthesis in the luminous fungal fruiting body.

We previously showed that luminous fungi share a common mechanism in bioluminescence, and identified hispidin as a luciferin precursor in Neonothopanus nambi mycelium. Here we showed the presence of hispidin as a bioluminescent active compound at 25-1000 pmol g-1 in the fruiting bodies of Mycena chlorophos, Omphalotus japonicus, and Neonothopanus gardneri. These results suggest that luminous mushrooms contain hispidin as a luciferin precursor. We also found that non-luminous "young" fruiting bodies exhibited luminescence by hispidin treatment. Furthermore, we observed a gradual luminescence enhancement of the cell-free fruiting body extract by the addition of hispidin biosynthetic components, namely caffeic acid, ATP and malonyl-CoA. These findings suggest that continuous weak glow of luminous mushrooms is regulated by slow recycling biosynthesis of hispidin.

Mechanism and color modulation of fungal bioluminescence.

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Genetic Organization of Anabaenopeptin and Spumigin Biosynthetic Gene Clusters in the Cyanobacterium Sphaerospermopsis torques-reginae ITEP-024.

Cyanobacteria produce a broad range of natural products, many of which are potent protease inhibitors. Biosynthetic gene clusters encoding the production of novel protease inhibitors belonging to the spumigin and anabaenopeptin family of nonribosomal peptides were identified in the genome of the bloom-forming cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. The genetic architecture and gene organization of both nonribosomal peptide biosynthetic clusters were compared in parallel with their chemical structure variations obtained by liquid chromatography (LC-MS/MS). The spumigin (spu) and anabaenopeptin (apt) gene clusters are colocated in the genomes of S. torques-reginae ITEP-024 and Nodularia spumigena CCY9414 and separated by a 12 kb region containing genes encoding a patatin-like phospholipase, l-homophenylalanine (l-Hph) biosynthetic enzymes, and four hypothetical proteins. hphABCD gene cluster encoding the production of l-Hph was linked to all eight apt gene clusters investigated here. We suggest that while the HphABCD enzymes are an integral part of the anabaenopeptin biosynthetic pathway, they provide substrates for the biosynthesis of both anabaenopeptins and spumigins. The organization of the spu and apt suggests a plausible model for the biosynthesis of the 4-(4-hydroxyphenyl)-2-acid (Hpoba) precursor of spumigin variants in S. torques-reginae ITEP-024 based on the acceptable substrates of HphABCD enzymes.

Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages.

Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques.

Growth and microcystin production of a Brazilian Microcystis aeruginosa strain (LTPNA 02) under different nutrient conditions

Cyanobacteria are prokaryotic and photosynthetic organisms, which can produce a wide range of bioactive compounds with different properties; including a variety of toxic compounds, also known as cyanotoxins. In this work, we describe the isolation of seven cyanobacterial strains from two reservoirs in São Paulo State, Brazil. Seven different chemical variants of microcystins (MC-RR, MC-LR, MC-YR, MC-LF, MC-LW, and two demethylated variants, dm-MC-RR and dm-MC-LR) were detected in three of the ten isolated strains. One particular Microcystis aeruginosa strain (LTPNA 02) was chosen to evaluate its growth by cell count, and its toxin production under seven different nutritional regimes. We observed different growth behaviors in the logarithmic growth period for only three experiments (p < 0.05). The total growth analysis identified four experiments as different from the control (p < 0.01). Three microcystin variants (MC-RR, MC-LR and MC-YR) were quantified by liquid chromatography-tandem mass spectrometry. At the experimental end, the toxin content was unchanged when comparing cell growth in ASM-1 (N:P = 1), MLA and BG-11 (N:P = 10) medium. In all other experiments, the lowest microcystin production was observed from cells grown in Bold 3N medium during the exponential growth phase. The highest microcystin content was observed in cultures using BG-11(N:P = 100) medium.

Biosynthesis of N,N-dimethyltryptamine (DMT) in a melanoma cell line and its metabolization by peroxidases.

Tryptophan (TRP) is essential for many physiological processes, and its metabolism changes in some diseases such as infection and cancer. The most studied aspects of TRP metabolism are the kynurenine and serotonin pathways. A minor metabolic route, tryptamine and N,N-dimethyltryptamine (DMT) biosynthesis, has received far less attention, probably because of the very low amounts of these compounds detected only in some tissues, which has led them to be collectively considered as trace amines. In a previous study, we showed a metabolic interrelationship for TRP in melanoma cell lines. Here, we identified DMT and N,N-dimethyl-N-formyl-kynuramine (DMFK) in the supernatant of cultured SK-Mel-147 cells. Furthermore, when we added DMT to the cell culture, we found hydroxy-DMT (OH-DMT) and indole acetic acid (IAA) in the cell supernatant at 24 h. We found that SK-Mel-147 cells expressed mRNA for myeloperoxidase (MPO) and also had peroxidase activity. We further found that DMT oxidation was catalyzed by peroxidases. DMT oxidation by horseradish peroxidase, H2O2 and MPO from PMA-activated neutrophils produced DMFK, N,N-dimethyl-kynuramine (DMK) and OH-DMT. Oxidation of DMT by peroxidases apparently uses the common peroxidase cycle involving the native enzyme, compound I and compound II. In conclusion, this study describes a possible alternative metabolic pathway for DMT involving peroxidases that has not previously been described in humans and identifies DMT and metabolites in a melanoma cell line. The extension of these findings to other cell types and the biological effects of DMT and its metabolites on cell proliferation and function are key questions for future studies.

Effects of a cyanobacterial extract containing-anatoxin-a(s) on the cardiac rhythm of Leurolestes circunvagans

This work presents the effects of an anatoxin-a(s)-containing extract on a cockroach semi-isolated heart preparation and the results supporting the extract’s biological activity on acetylcholinesterase (purified from ell). The presence of the toxin in cyanobacterial strains Anabaena spiroides (ITEP-024, ITEP-025 and ITEP-026) isolated from the Tapacurá reservoir in Pernambuco, Brazil, was confirmed by means of liquid chromatography coupled to an ion-trap mass spectrometer. The anticholinesterase activity was assessed biochemically by the Ellman test and was confirmed by measuring the cockroach’s heart rate. The concentration of the extract containing the tested anatoxin-a(s) (antx-a(s)) (10, 16 and 100 μg.μL-1) inhibited the eel acetylcholinesterase (AChE) by more than 90%. The cockroach cardiac frequency increased by a maximum of about 20% within 29 min after the addition of 2.5x10³ μg of extract containing antxa (s).g-1 bw (n=9, p<0.05). Our results strongly indicate that antx-a(s) is capable of exerting biological effects on cockroach, indicating that more research might be conducted to determine its role in the environment, especially on insects.

Intriguing differences in the gas-phase dissociation behavior of protonated and deprotonated gonyautoxin epimers.

The aim of this study was to investigate the unusual gas-phase dissociation behavior of two epimer pairs of protonated gonyautoxins (GTX) following electrospray ionization in comparison to their deprotonated counterparts. The chemical structures of the investigated GTX1-4 variants vary in their substitution pattern at N-1 and the stereochemical orientation of the hydroxysulfate group at C-11 (11α for GTX1/2 versus 11ß for GTX3/4). The direct comparison of mass spectra in positive and negative ion modes illustrated two distinct features: first, an intriguing difference between protonated 11α and 11ß species, where 11α conformations exhibited almost complete dissociation of [M + H](+) ions via facile SO(3) elimination, while 11ß species remained mostly intact as [M + H](+); and second, the lack of such differences for the deprotonated counterparts. In this study, we propose an acid-catalyzed elimination mechanism from density functional theory calculations, initiated by a proton transfer of a guanidinium proton to the hydroxysulfate group with simultaneous SO(3) release, which is only possible for the 11α conformation based on intramolecular distances. The same mechanism explains the lack of a comparable SO(3) loss in the negative ion mode. CID experiments supported this proposed mechanism for GTX1 and GTX2. Computational modeling of product ions seen in the CID spectra of GTX3 and GTX4 established that the lowest energy dissociation pathway for the 11ß epimers is elimination of water with the possibility for further SO(3) release from the intermediate product. Experimental data for structurally analogous decarbamoyl gonyautoxins confirmed the evidence for the GTX compounds as well as the proposed elimination mechanisms.