84 results on '"Viviani VR"'
Search Results
2. Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.
- Author
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Amaral, DT, Arnoldi, FGC, Rosa, SP, and Viviani, VR
- Abstract
ABSTRACT Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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3. Copper-Catalyzed Three-Component 1,5-Carboamination of Vinylcyclopropanes.
- Author
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Popov AG, Viviani VR, Skumial P, Jefferson TL, Salman SG, Baxter HH, and Hull KL
- Abstract
The 1,5-copper-catalyzed carboamination of vinylcyclopropanes is presented. A carbon-centered radical, formed upon reduction of an alkyl halide by Cu(I), adds across the alkene of a vinylcyclopropane, triggering ring opening to generate a benzylic radical, which, finally, undergoes copper-mediated amination to afford a homoallylic amine. The reaction occurs with outstanding regio- and good to very good diastereoselectivities. The scope of the reaction is demonstrated with respect to all three components: alkyl halide, vinylcyclopropane, and amine nucleophile. A total of 38 examples are presented with an average yield of 60%.
- Published
- 2024
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4. Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties.
- Author
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Pelentir GF, Tomazini A, Bevilaqua VR, and Viviani VR
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- Hydrogen-Ion Concentration, Animals, Color, Metals, Heavy chemistry, Metals, Heavy metabolism, Mercury chemistry, Mercury metabolism, Cadmium chemistry, Cadmium metabolism, Luciferases, Firefly metabolism, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Fireflies enzymology, Histidine chemistry, Histidine metabolism, Mutagenesis, Site-Directed
- Abstract
Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg., (© 2024. The Author(s), under exclusive licence to the European Photochemistry Association, European Society for Photobiology.)
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- 2024
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5. The orange light emitting luciferase from the rare Euryopa clarindae adult railroadworm (Coleoptera:Phengodidae): structural/functional and evolutionary relationship with green and red emitting luciferases.
- Author
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Viviani VR, Benites GR, Souza DR, Pelentir GF, Reis RM, Bechara EJH, and Tomazini A
- Subjects
- Animals, Phylogeny, Luciferases genetics, Luciferases chemistry, Larva, Brazil, Luminescent Measurements, Luciferases, Firefly, Coleoptera
- Abstract
Railroadworms luciferases emit the widest range of bioluminescence colors among beetles, ranging from green to red, being model enzymes to investigate the structure and bioluminescence colors relationships. Only three active railroadworms luciferases from the larval stage have been cloned and investigated: the Phrixothrix hirtus head lanterns red-emitting luciferase (PhRE); the Phrixothrix vivianii lateral lanterns green emitting luciferases (PvGR) and the Phengodes sp. dorsal lanterns yellow-green emitting luciferase (Ph). No active luciferase emitting in the yellow-orange region, however, has been cloned yet. Here we report the cloning and characterization of the orange emitting luciferase from the adult males of a rare Brazilian Cerrado railroadworm, Euryopa clarindae, and the transcriptional identification of two isozymes from the Amazon forest Mastinomorphus sp. railroadworm. The luciferase of E. clarindae has 548 residues, emits orange bioluminescence (600 nm), and displays intermediate kinetic values [K
M(luciferin) = 50 µM, KM(ATP) ~ 170 µM] between those reported for green-emitting lateral lanterns and red emitting head lanterns luciferases. It displays 74-78% identity with the lateral lanterns luciferases of other railroadworms and 70% with the head lantern PhRE luciferase, and 96% with the larval Mastinomorphus sp. Mast-1, suggesting that this larva could be from the Euryopa genus. The phylogenetic analysis and kinetic/functional properties, place this orange-emitting enzyme as an intermediate form between the green-emitting lateral lanterns and red-emitting head lanterns luciferases. Major structural differences that could be associated with bioluminescence color determination are a relatively larger cavity size, and substitutions in the loops 223-235 and 311-316, especially N/C/T311, and their interactions which may help to close the bottom of LBS., (© 2023. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)- Published
- 2024
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6. Biophosphorescence in fluorescent millipedes (Diplopoda: Xystodesmidae) and its relationships with bioluminescence.
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Viviani VR
- Subjects
- Animals, Fluorescence, Luminescence, Pteridines, Hydrogen Peroxide, Arthropods
- Abstract
Three types of luminescence have been reported in living organisms: bioluminescence (BL), ultraweak chemiluminescence and biofluorescence (FL). In millipedes, both BL and FL have been reported in Motyxia sequoiae and related Xystodesmidae species. Noteworthy, when walking at night with a UV lantern at the Biological Station of Highlands, I found three blue-fluorescent millipedes (Deltotaria brimleii, Deltotoria sp and Euryus orestes) that also displayed phosphorescence after turning off the UV source. The phosphorescence of the cuticle was in the green region (λ
max = 525 nm). The phosphorescence remained associated with cuticle and pellets, but frozen fluorescent supernatants, also displayed phosphorescence. The fluorescent compounds extracted from the cuticles in water and methanol and separated by TLC, displayed fluorescence spectra similar to that of 6-pteridine carboxylic acid. In contrast to Motyxia sequoiae cuticle extracts, no bioluminescence was found in Deltatoria and Euryus extracts in the presence of MgATP, but weak green chemiluminescence was detected with H2 O2 and superoxide. The spectral overlapping of phosphorescence of these millipedes with the bioluminescence of Motyxia (~ 507 nm) and the intimate association of both types of luminescence with the cuticles, raises the possibility that bioluminescence in Motyxia may arise from chemiluminescence reactions preferentially generating triplet excited states instead of singlet states., (© 2023. The Author(s).)- Published
- 2023
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7. Looking into luciferin.
- Author
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Viviani VR
- Published
- 2023
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8. Effect of pH on the secondary structure and thermostability of beetle luciferases: structural origin of pH-insensitivity.
- Author
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Tomazini A, Carvalho M, Murakami MT, and Viviani VR
- Subjects
- Animals, Luciferases, Firefly metabolism, Amino Acid Sequence, Luciferases chemistry, Fireflies, Luminescent Measurements, Coleoptera metabolism
- Abstract
Beetle luciferases were classified into three functional groups: (1) pH-sensitive yellow-green-emitting (fireflies) which change the bioluminescence color to red at acidic pH, high temperatures and presence of heavy metals; (2) the pH-insensitive green-yellow-emitting (click beetles, railroad worms and firefly isozymes) which are not affected by these factors, and (3) pH-insensitive red-emitting. Although the pH-sensing site in firefly luciferases was recently identified, it is unclear why some luciferases are pH-insensitive despite the presence of some conserved pH-sensing residues. Through circular dichroism, we compared the secondary structural changes and unfolding temperature of luciferases of representatives of these three groups: (1) pH-sensitive green-yellow-emitting Macrolampis sp2 (Mac) and Amydetes vivianii (Amy) firefly luciferases; (2) the pH-insensitive green-emitting Pyrearinus termitilluminans larval click beetle (Pte) and Aspisoma lineatum (Al2) larval firefly luciferases, and (3) the pH-insensitive red-emitting Phrixotrix hirtus railroadworm (PxRE) luciferase. The most blue-shifted luciferases, independently of pH sensitivity, are thermally more stable at different pHs than the red-shifted ones. The pH-sensitive luciferases undergo increases of α-helices and thermal stability above pH 6. The pH-insensitive Pte luciferase secondary structure remains stable between pH 6 and 8, whereas the Al2 luciferase displays an increase of the β-sheet at pH 8. The PxRE luciferase also displays an increase of α-helices at pH 8. The results indicate that green-yellow emission in beetle luciferases can be attained by: (1) a structurally rigid scaffold which stabilizes a single closed active site conformation in the pH-insensitive luciferases, and (2) active site compaction above pH 7.0 in the more flexible pH-sensitive luciferases., (© 2023. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)
- Published
- 2023
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9. Author Correction: A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).
- Author
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Viviani VR, Silva JR, Amaral DT, Bevilaqua VR, Abdalla FC, Branchini BR, and Johnson CH
- Published
- 2023
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10. Genome analysis of Phrixothrix hirtus (Phengodidae) railroad worm shows the expansion of odorant-binding gene families and positive selection on morphogenesis and sex determination genes.
- Author
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Amaral DT, Mitani Y, Bonatelli IAS, Cerri R, Ohmiya Y, and Viviani VR
- Subjects
- Animals, Female, Phylogeny, DNA Transposable Elements, Odorants, Luciferases metabolism, Morphogenesis, Pheromones, Railroads, Coleoptera genetics, Coleoptera metabolism
- Abstract
Among bioluminescent beetles of the Elateroidea superfamily, Phengodidae is the third largest family, with 244 bioluminescent species distributed only in the Americas, but is still the least studied from the phylogenetic and evolutionary points of view. The railroad worm Phrixothrix hirtus is an essential biological model and symbolic species due to its bicolor bioluminescence, being the only organism that produces true red light among bioluminescent terrestrial species. Here, we performed partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing the first source of genomic information and a framework for comparative analyses of the bioluminescent system in Elateroidea. This is the largest genome described in the Elateroidea superfamily, with an estimated size of ∼3.4 Gb, displaying 32 % GC content, and 67 % transposable elements. Comparative genomic analyses showed a positive selection of genes and gene family expansion events of growth and morphogenesis gene products, which could be associated with the atypical anatomical development and morphogenesis found in paedomorphic females and underdeveloped males. We also observed gene family expansion among distinct odorant-binding receptors, which could be associated with the pheromone communication system typical of these beetles, and retrotransposable elements. Common genes putatively regulating bioluminescence production and control, including two luciferase genes corresponding to lateral lanterns green-emitting and head lanterns red-emitting luciferases with 7 exons and 6 introns, and genes potentially involved in luciferin biosynthesis were found, indicating that there are no clear differences about the presence or absence of gene families associated with bioluminescence in Elateroidea., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)
- Published
- 2023
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11. Spectral tuning of bioluminescence and visual sensitivity in males of Brazilian firefly species inhabiting dim light environments (Coleoptera: Elateroidea: Lampyridae).
- Author
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Lall AB, Viviani VR, and Ventura DF
- Subjects
- Animals, Male, Brazil, Coleoptera physiology, Fireflies
- Abstract
Bioluminescence in fireflies is essential for sexual communication, and each species has evolved a specific bioluminescence emission capable of being detected by its visual system. This spectral "tuning" between visual sensitivity and bioluminescent emission has been established in 14 species of North American fireflies inhabiting diverse photoecological niches. Here we extend that research to three Brazilian species. Macrolampis omissa inhabits the Cerrado (savannas), while Photinus sp1 and Pyrogaster moestus are often sympatric species inhabiting borders of mesophyll rain forests and secondary growth. P. moestus particularly favors humid areas of the forest. M. omissa and Photinus sp1 are twilight-active fireflies emitting yellow bioluminescence. P. moestus is a "twi-night" species emitting green bioluminescence. It initiates flashing at the end of twilight and continues activity into the night. The visual spectral sensitivity of dark-adapted compound eyes in these three species is similar, showing a maximum in the yellow-green wavelengths and a secondary peak in the near-UV, suggesting the presence of two receptors. The bioluminescence emission spectrum in each species is tuned to its yellow-green visual sensitivity peak. Green chromatic adaptation experiments on Photinus sp1 and P. moestus suggest the presence of a blue receptor. The presence of near-UV, blue, and long-wavelength receptors in the compound eyes would enable a trichromatic color vision in Brazilian firefly species active in dim illumination., (© 2022 Wiley Periodicals LLC.)
- Published
- 2023
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12. Editorial: Bioluminescent indicators and sensors for biomedicine and environmental analysis.
- Author
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Viviani VR
- Abstract
Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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- 2022
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13. Cloning and molecular properties of a novel luciferase from the Brazilian Bicellonycha lividipennis (Lampyridae: Photurinae) firefly: comparison with other firefly luciferases.
- Author
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Moreira AC, Amaral DT, Gabriel GVM, and Viviani VR
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- Animals, Brazil, Cloning, Molecular, Luciferases chemistry, Phylogeny, Fireflies genetics, Luciferases, Firefly chemistry, Luciferases, Firefly genetics
- Abstract
Several firefly luciferases eliciting light emission in the yellow-green range of the spectrum and with distinct kinetic properties have been already cloned, sequenced, and characterized. Some of them are currently being applied as analytical reagents and reporter genes for bioimaging and biosensors, and more recently as potential color tuning indicators of intracellular pH and toxic metals. They were cloned from the subfamilies Lampyrinae (Photinini: Photinus pyralis, Macrolampis sp2; Cratomorphini: Cratomorphus distinctus), Photurinae (Photuris pennsylvanica), Luciolinae (Luciola cruciata, L. lateralis, L. mingrelica, L. italica, Hotaria parvula), and Amydetinae (Amydetes vivianii) occurring in different parts of the world. The largest number has been cloned from fireflies occurring in Brazilian biomes. Taking advantage of the large biodiversity of fireflies occurring in the Brazilian Atlantic rainforest, here we report the cloning and characterization of a novel luciferase cDNA from the Photurinae subfamily, Bicellonycha lividipennis, which is a very common firefly in marshlands in Brazil. As expected, multialignements and phylogenetic analysis show that this luciferase clusters with Photuris pennsylvanica adult isozyme, and with other adult lantern firefly luciferases, in reasonable agreement with traditional phylogenetic analysis. The luciferase elicits light emission in the yellow-green region, has kinetics properties similar to other adult lantern firefly luciferases, including pH- and metal sensitivities, but displays a lower sensitivity to nickel, which is suggested to be caused by the natural substitution of H310Y., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)
- Published
- 2022
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14. Biosensing firefly luciferin synthesis in bacteria reveals a cysteine-dependent quinone detoxification route in Coleoptera.
- Author
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de Souza DR, Silva JR, Moreira A, and Viviani VR
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- Animals, Cysteine metabolism, Escherichia coli genetics, Escherichia coli metabolism, Fireflies genetics, Luciferases genetics, Luciferases metabolism, Luciferins, Luminescent Measurements, Saccharomycetales metabolism, Coleoptera metabolism, Firefly Luciferin metabolism, Luciferases, Firefly metabolism, Quinones metabolism
- Abstract
Luciferin biosynthetic origin and alternative biological functions during the evolution of beetles remain unknown. We have set up a bioluminescent sensing method for luciferin synthesis from cysteine and benzoquinone using E. coli and Pichia pastoris expressing the bright Amydetes vivianii firefly and P. termitilluminans click beetle luciferases. In the presence of D-cysteine and benzoquinone, intense bioluminescence is quickly produced, indicating the expected formation of D-luciferin. Starting with L-cysteine and benzoquinone, the bioluminescence is weaker and delayed, indicating that bacteria produce L-luciferin, and then racemize it to D-luciferin in the presence of endogenous esterases, CoA and luciferase. In bacteria the p-benzoquinone toxicity (I
C50 ~ 25 µM) is considerably reduced in the presence of cysteine, maintaining cell viability at 3.6 mM p-benzoquinone concomitantly with the formation of luciferin. Transcriptional analysis showed the presence of gene products involved with the sclerotization/tanning in the photogenic tissues, suggesting a possible link between these pathways and bioluminescence. The lack of two enzymes involved with the last steps of these pathways, indicate the possible accumulation of toxic quinone intermediates in the lanterns. These results and the abundance of cysteine producing enzymes suggest that luciferin first appeared as a detoxification byproduct of cysteine reaction with accumulated toxic quinone intermediates during the evolution of sclerotization/tanning in Coleoptera., (© 2022. The Author(s).)- Published
- 2022
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15. Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing.
- Author
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Viviani VR, Pelentir GF, and Bevilaqua VR
- Subjects
- Animals, Fireflies genetics, Hydrogen-Ion Concentration, Luciferases chemistry, Luciferases genetics, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Luminescent Measurements methods, Mammals, Mercury, Metals, Heavy chemistry
- Abstract
Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical purposes for over 5 decades. Under acidic conditions, high temperatures and the presence of heavy metals, they produce red light, a property that is called pH-sensitivity or pH-dependency. Despite the demand for physiological intracellular biosensors for pH and heavy metals, firefly luciferase pH and metal sensitivities were considered drawbacks in analytical assays. We first demonstrated that firefly luciferases and their pH and metal sensitivities can be harnessed to estimate intracellular pH variations and toxic metal concentrations through ratiometric analysis. Using Macrolampis sp2 firefly luciferase, the intracellular pH could be ratiometrically estimated in bacteria and then in mammalian cells. The luciferases of Macrolampis sp2 and Cratomorphus distinctus fireflies were also harnessed to ratiometrically estimate zinc, mercury and other toxic metal concentrations in the micromolar range. The temperature was also ratiometrically estimated using firefly luciferases. The identification and engineering of metal-binding sites have allowed the development of novel luciferases that are more specific to certain metals. The luciferase of the Amydetes viviani firefly was selected for its special sensitivity to cadmium and mercury, and for its stability at higher temperatures. These color-tuning luciferases can potentially be used with smartphones for hands-on field analysis of water contamination and biochemistry teaching assays. Thus, firefly luciferases are novel color-tuning sensors for intracellular pH and toxic metals. Furthermore, a single luciferase gene is potentially useful as a dual bioluminescent reporter to simultaneously report intracellular ATP and/or luciferase concentrations luminometrically, and pH or metal concentrations ratiometrically, providing a useful tool for real-time imaging of intracellular dynamics and stress.
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- 2022
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16. Synthesis of bioluminescent gold nanoparticle-luciferase hybrid systems for technological applications.
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Belleti E, Bevilaqua VR, Brito AMM, Modesto DA, Lanfredi AJC, Viviani VR, and Nantes-Cardoso IL
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- Animals, Gold, Luciferases genetics, Luminescence, Luminescent Measurements, Coleoptera, Metal Nanoparticles
- Abstract
Bioluminescent gold nanoparticles (AuNPs) were synthesized in situ using dithiol-terminated polyethylene glycol (PEG(SH)
2 ) as reducer and stabilizing agents. Hybrid Au/F3 O4 nanoparticles were also produced in a variation of synthesis, and both types of nanostructures had the polymer capping replaced by L-cysteine (Cys). The four types of nanoparticles, PEG(SH)2 AuNPs, PEG(SH)2 Au/F3 O4 NPs, CysAuNPs, and CysAu/F3 O4 NPs were associated with purified recombinant Pyrearinus termitilluminans green emitting click beetle luciferase (PyLuc) and Phrixotrix hirtus (RELuc) red-emitting railroad worm luciferase. Enzyme association with PEG(SH)2 was also investigated as a control. Luciferases were chosen because they catalyze bioluminescent reactions used in a wide range of bioanalytical applications, including ATP assays, gene reporting, high-throughput screening, bioluminescence imaging, biosensors and other bioluminescence-based assays. The immobilization of PyLuc and RELuc promoted partial suppression of the enzyme luminescence activity in a functionalization-dependent way. Association of PyLuc and RELuc with AuNPs increased the enzyme operational stability in relation to the free enzyme, as evidenced by the luminescence intensity from 0 to 7 h after substrate addition. The stability of the immobilized enzymes was also functionalization-dependent and the association with CysAuNPs was the condition that combined more sustained luminescent activity with a low degree of luminescence quenching. The higher enzymatic stability and sustained luminescence of luciferases associated with nanoparticles may improve the applicability of bioluminescence for bioimaging and biosensing purposes., (© 2021. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)- Published
- 2021
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17. A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays.
- Author
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Viviani VR, Silva JR, and Ho PL
- Abstract
Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t
1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1-250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5-250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Viviani, Silva and Ho.)- Published
- 2021
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18. RNA-Seq analysis of the blue light-emitting Orfelia fultoni (Diptera: Keroplatidae) suggest photoecological adaptations at the molecular level.
- Author
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Amaral DT, Johnson CH, and Viviani VR
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- Animals, Diptera radiation effects, Ecosystem, Insect Proteins genetics, Light, Luminescent Proteins genetics, Proteome analysis, Adaptation, Physiological, Diptera genetics, Insect Proteins metabolism, Luminescence, Luminescent Proteins metabolism, RNA-Seq methods, Transcriptome radiation effects
- Abstract
Bioluminescence in Diptera is found in the Keroplatidae family, within Arachnocampininae and Keroplatinae subfamilies, with reported occurrences in Oceania, Eurasia, and Americas. Larvae of Orfelia fultoni, which inhabit stream banks in the Appalachian Mountains, emit the bluest bioluminescence among insects, using it for prey attraction, similarly to Arachnocampa spp. Although bioluminescence has a similar prey attraction function, the systems of Arachonocampininae and Keroplatinae subfamilies are morphologically/biochemically distinct, indicating different evolutionary origins. To identify the possible coding genes associated with physiological control, ecological adaptations, and origin/evolution of bioluminescence in the Keroplatinae subfamily, we performed the RNA-Seq analysis of O. fultoni larvae during day and night and compared it with the transcriptomes of Arachnocampa luminosa, and reanalyzed the previously published proteomic data of O. fultoni against the RNA-Seq dataset. The abundance of chaperones/heat-shock and hexamerin gene products at night and in luciferase enriched fractions supports their possible association and participation in bioluminescence. The low diversity of copies/families of opsins indicate a simpler visual system in O. fultoni. Noteworthy, gene products associated with silk protein biosynthesis in Orfelia were more similar to Lepidoptera than to the Arachnocampa, indicating that, similarly to the bioluminescent systems, at some point, the biochemical apparatus for web construction may have evolved independently in Orfelia and Arachnocampa., (Copyright © 2021. Published by Elsevier Inc.)
- Published
- 2021
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19. Selective inhibition of Zophobas morio (Coleoptera: Tenebrionidae) luciferase-like enzyme luminescence by diclofenac and potential suitability for light-off biosensing.
- Author
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Carvalho MC, Tomazini A, Prado RA, and Viviani VR
- Subjects
- Amino Acid Sequence, Animals, Diclofenac, Firefly Luciferin, Kinetics, Luciferases genetics, Luciferases metabolism, Luminescence, Coleoptera
- Abstract
The accumulation of toxic carboxylic compounds may cause severe effects on the environment and living organisms. A luciferase-like enzyme, previously cloned from the Malpighian tubules of the non-luminescent Zophobas morio mealworm, displays thioesterification activity with a wide range of carboxylic substrates, and produces weak red luminescence in the presence of ATP and firefly d-luciferin, a xenobiotic for this organism. To better investigate the function of this enzyme in carboxylic xenobiotic detoxification, we analyzed the inhibitory effect of different xenobiotic carboxylic acids on the luminescence activity of this enzyme, including environmental pollutants and pharmaceutical compounds. Noteworthy, the anti-inflammatory drug diclofenac severely inhibited this luciferase-like enzyme luminescence activity, both in in vitro (IC
50 20 μM) and in vivo in bacterial cells assays, when compared with other beetle luciferases. Similar results were obtained with its brighter I327S mutant. Kinetic analysis of diclofenac's effect on luminescence activity indicated mixed-type inhibition for both ATP and d-luciferin. Modelling studies showed five potential binding sites for diclofenac, including the coenzyme A binding site, which showed one of the highest binding constant. Taken together, these results raise the possibility of using this luciferase-like enzyme for the development of novel whole-cell luminescent biosensors for diclofenac and similar drugs., (© 2020 John Wiley & Sons, Ltd.)- Published
- 2021
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20. Influence of the C-terminal domain on the bioluminescence activity and color determination in green and red emitting beetle luciferases and luciferase-like enzyme.
- Author
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Bevilaqua VR, Carvalho MC, Pelentir GF, Tomazini A, Murakami M, and Viviani VR
- Subjects
- Amino Acid Sequence, Animals, Benzothiazoles chemistry, Benzothiazoles metabolism, Binding Sites, Coleoptera enzymology, Insect Proteins chemistry, Insect Proteins genetics, Kinetics, Luciferases chemistry, Luciferases genetics, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Luciferases, Firefly metabolism, Luminescent Measurements, Molecular Docking Simulation, Mutagenesis, Protein Domains, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Insect Proteins metabolism, Luciferases metabolism
- Abstract
Beetle luciferases catalyze the bioluminescent oxidation of D-luciferin, producing bioluminescence colors ranging from green to red, using two catalytic steps: adenylation of D-luciferin to produce D-luciferyl-adenylate and PPi, and oxidation of D-luciferyl-adenylate, yielding AMP, CO
2 , and excited oxyluciferin, the emitter. Luciferases and CoA-ligases display a similar fold, with a large N-terminal domain, and a small C-terminal domain which undergoes rotation, closing the active site and promoting both adenylation and oxidative reactions. The effect of C-terminal domain deletion was already investigated for Photinus pyralis firefly luciferase, resulting in a red-emitting mutant with severely impacted luminescence activity. However, the contribution of C-terminal in the bioluminescence activities and colors of other beetle luciferases and related ancestral luciferases were not investigated yet. Here we compared the effects of the C-terminal domain deletion on green-emitting luciferases of Pyrearinus termitilluminans (Pte) click beetle and Phrixothrix vivianii railroadworm, and on the red-emitting luciferase of Phrixothrix hirtus railroadworm and luciferase-like enzyme of Zophobas morio. In all cases, the domain deletion severely impacted the overall bioluminescence activities and, slightly less, the oxidative activities, and usually red-shifted the bioluminescence colors. The results support the involvement of the C-terminal in shielding the active site from the solvent during the light emitting step. However, in Pte luciferase, the deletion caused only a 10 nm red-shift, indicating a distinctive active site which remains more shielded, independently of the C'-terminal. Altogether, the results confirm the main contribution of the C-terminal for the catalysis of the adenylation reaction and for active site shielding during the light emitting step.- Published
- 2021
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21. A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6'-Amino-Analogs for Bioimaging Purposes.
- Author
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Viviani VR, Bevilaqua VR, de Souza DR, Pelentir GF, Kakiuchi M, and Hirano T
- Subjects
- Amino Acid Substitution, Animals, Enzyme Stability, Firefly Luciferin chemistry, Kinetics, Luciferases metabolism, Mutagenesis, Site-Directed, Mutation, Spectrum Analysis, Structure-Activity Relationship, Thermodynamics, Light, Luciferases genetics, Luminescent Agents chemistry, Luminescent Measurements methods, Molecular Imaging methods
- Abstract
Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6'-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636-655 nm. Among them, the combination of PxRE-R215K mutant with 6'-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.
- Published
- 2020
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22. Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.
- Author
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Carvalho MC, Tomazini A, Amaral DT, Murakami MT, and Viviani VR
- Subjects
- Animals, Hydrogen-Ion Concentration, Reproduction, Fat Body enzymology, Fireflies enzymology, Luciferases, Firefly metabolism
- Abstract
Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.
- Published
- 2020
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23. Role of E270 in pH- and metal-sensitivities of firefly luciferases.
- Author
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Viviani VR, Pelentir GF, Oliveira G, Tomazini A, and Bevilaqua VR
- Subjects
- Animals, Fireflies, Hydrogen-Ion Concentration, Kinetics, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Metals, Heavy chemistry, Mutagenesis, Site-Directed, Luciferases, Firefly metabolism, Metals, Heavy metabolism
- Abstract
Firefly luciferases display a typical change in bioluminescence color to red at acidic pH, high temperatures and in the presence of heavy metals. Recently, the proton and metal sensing site responsible for the pH-sensitivity of firefly luciferases, which involves the salt bridges between E311-R337 and H310-E354, was identified. However, it is unclear what other residues contribute to the distinct degrees of pH-sensitivity observed in other firefly luciferases. A multialignment of primary structures of a large set of pH-sensitive and pH-insensitive beetle luciferases showed that the conserved E270 among adult firefly luciferases is substituted by Gly (railroad worms)/Gln (click-beetles) in pH-insensitive ones. Site-directed mutagenesis studies using Macrolampis sp2 and Amydetes vivianii firefly luciferases indeed showed that E270 is important for the pH-dependent activity and spectral profiles: the substitution E270A/G drastically decreases the spectral pH-sensitivity, and extends the activity profile above pH 9.0. These mutations also decrease the sensitivity to metals such as zinc, mercury and cadmium. Modelling studies showed that the residue E270 is located in a three-glutamate motif (269EEE271) at the N-terminal of α-helix-10. The results suggest that at acidic pH, the protonation of E270 carboxylate may extend a turn of the helix at the N-terminal, misaligning the pH-sensor and luciferin phenolate binding site residues: S286, I288 and E311. In contrast, the substitution of E270A/G may unwind a turn of the α-helix-10, indirectly increasing the interaction of the pH-sensor and other residues at the bottom of the luciferin binding site, stabilizing the green light emitting conformation.
- Published
- 2020
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24. A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).
- Author
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Viviani VR, Silva JR, Amaral DT, Bevilaqua VR, Abdalla FC, Branchini BR, and Johnson CH
- Subjects
- Animals, Chromatography, Ion Exchange, Diptera enzymology, Firefly Luciferin chemistry, Firefly Luciferin isolation & purification, Gene Expression Profiling, Luciferases chemistry, Luciferases isolation & purification, Luminescent Measurements, Mitochondria enzymology, Mitochondria metabolism, Spectrometry, Fluorescence, Diptera metabolism, Firefly Luciferin metabolism, Luciferases metabolism
- Abstract
Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.
- Published
- 2020
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25. Morphology of immature stages of Pyrearinus pumilus (Candèze, 1863 (Coleoptera: Elateridae: Pyrophorini): the click beetle responsible for the luminous canga caves in the state of Pará, Brazil.
- Author
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Rosa SP, Mariano RDR, Viviani VR, and Costa C
- Subjects
- Animals, Brazil, Larva, Male, Pupa, Coleoptera
- Abstract
The Pyrearinus pumilus species group from South America includes luminescent click beetles (Agrypninae: Pyrophorini) associated with the phenomena of "luminescent termite mounds" and "luminous canga caves". The latter was recently reported in the state of Pará, Brazil. This group includes six species based on the morphology of adults, of which two have immature stages already described. In this work we present the morphology and biological aspects of mature larva and pupa of Pyrearinus pumilus (Candèze, 1863), from the canga caves. Moreover, we provide a key and illustrations for identification of male adults and the known larvae. Our study shows that: (1) morphological characters of immatures support the close relationship of P. pumilus with their allies in the P. pumilus species group; (2) the traits of the known larvae of the P. pumilus species group are reliable for species identification.
- Published
- 2020
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26. Temperature effect on the bioluminescence spectra of firefly luciferases: potential applicability for ratiometric biosensing of temperature and pH.
- Author
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Oliveira G and Viviani VR
- Subjects
- Animals, Biosensing Techniques methods, Coleoptera metabolism, Hydrogen-Ion Concentration, Luciferases, Firefly genetics, Luciferases, Firefly metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Temperature, Luciferases, Firefly chemistry, Luminescent Measurements methods
- Abstract
Bioluminescence spectra of firefly luciferases are affected by pH, heavy metals and high temperatures. Previously, we compared the effect of pH and heavy metals on the bioluminescence spectra of different firefly luciferases and showed that such spectral sensitivity can be harnessed to ratiometrically estimate the pH inside cells and metal concentration. Here, we compared the effect of temperature on the spectral sensitivity of four firefly luciferases (Amydetes vivianii: 539 nm; Cratomorphus distinctus: 548 nm; Photinus pyralis: 558 nm and Macrolampis sp2: 594 nm) and investigated whether a ratiometric curve could be used to estimate temperature. The ratio of intensities of bioluminescence at two wavelengths (green and red) at different temperatures (5-35 °C) was determined. The results confirm that, in the case of pH-sensitive luciferases, the more blue-shifted the bioluminescence spectrum, the more thermostable the enzyme and the less sensitive the emission spectrum to temperature. An almost linear relationship between temperature and the ratio of bioluminescence intensities in the green and red region of the spectrum was found for the four luciferases: the more blue-shifted and less sensitive luciferases exhibit a smaller slope and the more red-shifted luciferases exhibit a steeper slope in the following order: Amy < Crt < Ppy < Mac. This relationship offers the possibility of using firefly luciferases as ratiometric indicators of temperature and may allow the compensation of the effect of temperature in the ratiometric analysis of intracellular pH and heavy metal concentration for each enzyme.
- Published
- 2019
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27. Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae) is the first record of a bioluminescent fungus-gnat in South America.
- Author
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Falaschi RL, Amaral DT, Santos I, Domingos AHR, Johnson GA, Martins AGS, Viroomal IB, Pompéia SL, Mirza JD, Oliveira AG, Bechara EJH, Viviani VR, and Stevani CV
- Subjects
- Animals, Brazil, Luminescence, Nematocera anatomy & histology, Nematocera genetics, Phylogeny, Larva anatomy & histology, Larva genetics, Larva physiology, Nematocera physiology
- Abstract
Blue shining fungus gnats (Diptera) had been long reported in the Waitomo caves of New Zealand (Arachnocampa luminosa Skuse), in stream banks of the American Appalachian Mountains (Orfelia fultoni Fisher) in 1939 and in true spore eating Eurasiatic Keroplatus Bosc species. This current report observes that similar blue light emitting gnat larvae also occur nearby the Betary river in the buffer zone of High Ribeira River State Park (PETAR) in the Atlantic Forest of Brazil, where the larvae were found when on fallen branches or trunks enveloped in their own secreted silk. The new species is named Neoceroplatus betaryiensis nov. sp. (Diptera: Keroplatidae: Keroplatinae: Keroplatini) based on a morphological analysis. Neoceroplatus betaryiensis nov. sp. larvae emit blue bioluminescence that can be seen from their last abdominal segment and from two photophores located laterally on the first thoracic segment. When touched, the larvae can actively stop its luminescence, which returns when it is no longer being agitated. The in vitro bioluminescence spectrum of N. betaryiensis nov. sp. peaks at 472 nm, and cross-reactivity of hot and cold extracts with the luciferin-luciferase from Orfelia fultoni indicate significant similarity in both enzyme and substrate of the two species, and that the bioluminescence system in the subfamily Keroplatinae is conserved.
- Published
- 2019
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28. A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays.
- Author
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Pelentir GF, Bevilaqua VR, and Viviani VR
- Subjects
- Animals, Cadmium chemistry, Fluorescence, Hydrogen-Ion Concentration, Kinetics, Luciferases, Firefly chemistry, Metals, Heavy chemistry, Adenosine Triphosphate analysis, Biosensing Techniques, Cadmium metabolism, Fireflies enzymology, Luciferases, Firefly metabolism, Metals, Heavy metabolism, Temperature
- Abstract
Firefly luciferases have been widely used for bioanalytical purposes during the last 5 decades. They usually emit yellow-green bioluminescence and are pH-sensitive, displaying a color change to red at acidic pH and higher temperature and in the presence of heavy metals. Besides the usual applications as bioanalytical reagents and as reporter genes, firefly luciferases' pH- and metal-sensitivities have been recently harnessed for intracellular metal and pH biosensing. Previously we cloned the luciferase of the Brazilian Amydetes vivianii firefly which displays the most blue-shifted color among known firefly luciferases. Here we purified it, characterized and investigated the kinetic properties and the pH, metal and thermal sensitivities of this firefly luciferase. This luciferase displays the lowest reported K
M for ATP, the highest catalytic efficiencies, and the highest thermostability among the studied recombinant beetle luciferases, making this enzyme and its cDNA an ideal reagent for sensitive ATP assays and reporter gene. The blue-shifted spectrum, higher thermostability, lower pH- and thermal-sensitivities and protein fluorescence studies indicate a more rigid active site during light emission. This enzyme displays an unmatched selective spectral sensitivity for cadmium and mercury, making it a promising ratiometric indicator of such toxic metals. Finally, the weaker thermal-sensitivity compared to other firefly luciferases makes this enzyme a better ratiometric pH indicator at temperatures above 30 °C, suitable for mammalian cell assays.- Published
- 2019
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29. Phrixotrix luciferase and 6'-aminoluciferins reveal a larger luciferin phenolate binding site and provide novel far-red combinations for bioimaging purposes.
- Author
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Bevilaqua VR, Matsuhashi T, Oliveira G, Oliveira PSL, Hirano T, and Viviani VR
- Subjects
- Amino Acid Sequence, Animals, Binding Sites genetics, Coleoptera genetics, Color, Firefly Luciferin chemistry, Insect Proteins chemistry, Insect Proteins genetics, Kinetics, Luciferases chemistry, Luciferases genetics, Luminescent Proteins chemistry, Luminescent Proteins genetics, Luminescent Proteins metabolism, Molecular Structure, Mutagenesis, Site-Directed, Sequence Homology, Amino Acid, Coleoptera metabolism, Firefly Luciferin metabolism, Insect Proteins metabolism, Luciferases metabolism, Luminescence, Luminescent Measurements methods
- Abstract
How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6'-substituted aminoluciferin analogues (6'-morpholino- and 6'-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the K
M and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6'-morpholino- (634 nm) and 6'-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6'-aminoluciferins provide potential far-red combinations for bioimaging applications.- Published
- 2019
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30. Phylogenomic analyses and divergence time estimation of Elateroidea (Coleoptera) based on RNA-Seq data.
- Author
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Amaral DT, Bonatelli IAS, Cerri R, and Viviani VR
- Subjects
- Animals, Biological Evolution, Genomics, Luminescence, RNA-Seq, Transcriptome, Coleoptera genetics, Phylogeny
- Abstract
Bioluminescence, the emission of visible light in a living organism, is an intriguing phenomenon observed in different species and environments. In terrestrial organisms, the bioluminescence is observed mainly in beetles of the Elateroidea superfamily (Coleoptera). Several phylogenetic studies have been used different strategies to propose a scenario for the origin and evolution of the bioluminescence within this group, however some of them showed incongruences, mainly about the relationship of the bioluminescent families. In order to increase the number of molecular markers available for Elateroidea species and to propose a more accurate phylogeny, with high supported topology, we employed the Next-Generation Sequencing (NGS) methodology to perform the RNA-Seq analysis of luminescent (Elateridae, Phengodidae, Rhagophthalmidae, and Lampyridae) and non-luminescent (Cantharidae) species of Neotropical beetles. We used the RNA-Seq data to construct a calibrated phylogeny of Elateroidea superfamily using a large number of nuclear molecular markers. The results indicate Lampyridae and Phengodidae/Rhagophthalmidae as sister-groups, suggesting that the bioluminescence evolved later in Elateridae than other families (Lampyridae, Phengodidae, and Rhagophthalmidae), and indicating the Upper Cretaceous as the period for the main diversification of Elateroidea bioluminescent species., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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31. Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells.
- Author
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Gabriel GVM, Yasuno R, Mitani Y, Ohmiya Y, and Viviani VR
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Fireflies, Hydrogen-Ion Concentration, Organelles chemistry, Biosensing Techniques, Luciferases, Firefly metabolism, Luminescent Measurements, Organelles metabolism
- Abstract
Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.
- Published
- 2019
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32. RNA-Seq analysis of the bioluminescent and non-bioluminescent species of Elateridae (Coleoptera): Comparison to others photogenic and non-photogenic tissues of Elateroidea species.
- Author
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Amaral DT, Silva JR, and Viviani VR
- Subjects
- Animals, Fat Body metabolism, Luminescence, Coleoptera genetics, Genes, Insect, Sequence Analysis, RNA methods
- Published
- 2019
- Full Text
- View/download PDF
33. Comparison of the Malpighian tubules and fat body transcriptional profiles of Zophobas morio larvae (Coleoptera: Tenebrionidae).
- Author
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Silva JR, Amaral DT, and Viviani VR
- Subjects
- Amino Acid Sequence, Animals, Coleoptera growth & development, Insect Proteins chemistry, Insect Proteins genetics, RNA, Messenger genetics, Sequence Homology, Amino Acid, Coleoptera genetics, Fat Body metabolism, Gene Expression Profiling, Genes, Insect, Larva metabolism, Malpighian Tubules metabolism, Transcription, Genetic
- Abstract
The Malpighian tubules in insects play an essential role in osmoregulation, through the transport of ions during excretion, whereas the fat body is usually associated with the intermediary metabolism. The tubules also are involved in excretion of organic solutes and xenobiotics. However, with the exception of a preliminary transcriptional survey of the Zophobas morio (Tenebrionidae) larval tubules, there are no detailed transcriptional analysis of this organ in Coleoptera. A luciferase-like enzyme that displays weak luminescence activity in the presence of firefly D-luciferin and ATP was cloned from the tubules of Z. morio larvae. In order to better understand the molecular physiology of Malpighian tubules and fat body in Coleoptera larvae, and to investigate the occurrence and functions of AMP-CoA ligases in these tissues, we performed a comparative transcriptional analysis of these tissues using Z. morio giant-mealworms. As expected, the tubules displayed organic and inorganic transporters, xenobiotic metabolism enzymes, V-ATPases, channels, and pumps. The fat body showed proteins that are synthesized in this tissue and secreted to the hemolymph, as well as enzymes involved in lipid and carbohydrate metabolism. These tissues are also involved in common pathways, such as nitrogen metabolism to degradation/excretion, eye pigments biosynthesis, immunity, and detoxification. The presence of coumarate-CoA ligase-like enzymes in these tissues suggest their involvement in the degradation of coumaric acid derivatives obtained from the diet, or alternatively, in the biosynthesis of compounds structurally related to coumaric acids such as eye pigments. Our results confirm to the physiological versatility of tubules and fat body in larval Coleoptera., (Copyright © 2018. Published by Elsevier Inc.)
- Published
- 2019
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34. The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases.
- Author
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Viviani VR, Gabriel GVM, Bevilaqua VR, Simões AF, Hirano T, and Lopes-de-Oliveira PS
- Abstract
Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.
- Published
- 2018
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35. Orfelia-type luciferin and its associated storage protein in the non-luminescent cave worm Neoditomyia sp. (Diptera: Keroplatidae) from the Atlantic rainforest: biological and evolutionary implications.
- Author
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Viviani VR, Amaral DT, Bevilaqua VR, and Falaschi RL
- Subjects
- Animals, Biological Evolution, Diptera genetics, Larva chemistry, Larva genetics, Luminescence, Phylogeny, Rainforest, Diptera chemistry, Firefly Luciferin analysis, Luminescent Agents analysis
- Abstract
Bioluminescence in Diptera is found in the family Keroplatidae, in the glowworms of the genera Arachnocampa, Orfelia and Keroplatus. Despite belonging to the same family, Arachnocampa spp. and Orfelia fultoni display morphologically and biochemically distinct bioluminescence systems: Arachnocampa spp. produce light by the terminal ends of Malpighian tubules using ATP, a luciferin and a luciferase, whereas Orfelia fultoni produces light by translucent areas associated with rows of black bodies in the anterior and posterior parts of the body, using a 140 kDa luciferase and a luciferin which do not cross-react with the Arachnocampa luciferin-luciferase system, and a substrate binding fraction (SBF) which apparently releases luciferin in the presence of reductants. While several other keroplatids are not luminescent, we recently discovered a non-luminescent web-constructing keroplatid larva living in the roofs of caves in the Atlantic rainforest in Brazil, which noteworthily has a compound with Orfelia luciferin-like activity and its associated binding protein (SBF). Both the Neoditomyia luciferin-like compound and SBF cross-react with purified Orfelia luciferase to produce light in the same blue region of the Orfelia luciferin-luciferase system (479 nm). We also checked for the presence of Orfelia-type luciferin in Arachnocampa luminosa and Aedes aegytpi larval bodies, but no traces were found. Molecular studies indicate that Neoditomyia sp. is phylogenetically closer to Keroplatus and Orfelia than to Arachnocampa species. The presence of luciferin and its associated binding protein in this non-bioluminescent keroplatid larva indicates that luciferin may display another important biochemical function in keroplatid larvae and suggests that bioluminescence could be a recently evolved trait in Keroplatidae.
- Published
- 2018
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36. Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours.
- Author
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Oliveira G and Viviani VR
- Subjects
- Animals, Brazil, Color, Enzyme Stability, Firefly Luciferin chemistry, Hot Temperature, Insect Proteins genetics, Insect Proteins metabolism, Kinetics, Luciferases genetics, Luciferases metabolism, Luciferases, Firefly chemistry, Luciferases, Firefly metabolism, Luminescence, Luminescent Measurements, Coleoptera enzymology, Insect Proteins chemistry, Luciferases chemistry
- Abstract
Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue-shifted examples in each family, confirming the trend that the most blue-shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases)., (Copyright © 2017 John Wiley & Sons, Ltd.)
- Published
- 2018
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37. Revisiting Coleoptera a + T-rich region: structural conservation, phylogenetic and phylogeographic approaches in mitochondrial control region of bioluminescent Elateridae species (Coleoptera).
- Author
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Amaral DT, Mitani Y, Oliveira G, Ohmiya Y, and Viviani VR
- Subjects
- Animals, Base Sequence, Coleoptera genetics, Conserved Sequence, DNA, Mitochondrial genetics, Evolution, Molecular, Genetic Variation, Phylogeny, Phylogeography, Coleoptera classification, DNA, Mitochondrial chemistry, Mitochondria genetics, Sequence Analysis, DNA methods
- Abstract
The control region (CR) or A + T-rich region in Coleoptera mt genome is poorly characterized, including the Elateroidea bioluminescent species. Here, we provided the first attempt to characterize and compare the structure and organization of the CR of different species within Elateridae. We also revisited some sequenced Coleoptera CR and observed consensus T-stretches, non-conserved sequences near the stem-loop and unusual inner tRNAs-like sequences. All these features are probably involved in the replication start of the mt genome. The phylogenetic relationships in Elateridae bioluminescent groups using partial sequence of CR showed the monophyly of Pyrearinus pumilus group and Pyrearinus as a polyphyletic genus, corroborating our previous results. The wider genetic variation obtained by CR analysis could separate two different lineages that occur within P. termitilluminans populations. In Elateridae, the CR exhibited high polymorphism within and between populations, which was also observed in other Coleoptera species, suggesting that the CR could be described as a suitable molecular marker to be applied in phylogenetic and phylogeographic studies.
- Published
- 2017
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- View/download PDF
38. Spectroscopic Properties of Amine-substituted Analogues of Firefly Luciferin and Oxyluciferin.
- Author
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Kakiuchi M, Ito S, Yamaji M, Viviani VR, Maki S, and Hirano T
- Subjects
- Firefly Luciferin analogs & derivatives, Luciferases, Firefly chemistry, Luminescence, Proton Magnetic Resonance Spectroscopy, Solvents chemistry, Spectrometry, Mass, Electrospray Ionization, Amines chemistry, Firefly Luciferin chemistry, Indoles chemistry, Pyrazines chemistry, Spectrometry, Fluorescence methods, Spectrophotometry, Ultraviolet methods
- Abstract
Spectroscopic and photophysical properties of firefly luciferin and oxyluciferin analogues with an amine substituent (NH
2 , NHMe and NMe2 ) at the C6' position were studied based on absorption and fluorescence measurements. Their π-electronic properties were investigated by DFT and TD-DFT calculations. These compounds showed fluorescence solvatochromism with good quantum yields. An increase in the electron-donating strength of the substituent led to the bathochromic shift of the fluorescence maximum. The fluorescence maxima of the luciferin analogues and the corresponding oxyluciferin analogues in a solvent were well correlated with each other. Based on the obtained data, the polarity of a luciferase active site was explained. As a result, the maximum wavelength of bioluminescence for a luciferin analogue was readily predicted by measuring the photoluminescence of the luciferin analogue in place of that of the corresponding oxyluciferin analogue., (© 2016 The American Society of Photobiology.)- Published
- 2017
- Full Text
- View/download PDF
39. Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.
- Author
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Gabriel GV and Viviani VR
- Subjects
- Animals, Cations, Divalent, Cloning, Molecular, Escherichia coli genetics, Escherichia coli metabolism, Fireflies enzymology, Fireflies genetics, Fireflies metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Glutamic Acid chemistry, Glutamic Acid metabolism, Histidine chemistry, Histidine metabolism, Kinetics, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Luminescence, Mutagenesis, Site-Directed, Protein Engineering, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Biosensing Techniques methods, Luciferases, Firefly metabolism, Luminescent Measurements methods, Mercury analysis, Nickel analysis, Zinc analysis
- Abstract
Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.
- Published
- 2016
- Full Text
- View/download PDF
40. A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.
- Author
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Amaral DT, Oliveira G, Silva JR, and Viviani VR
- Subjects
- Animals, Brazil, Luciferases genetics, Luciferases metabolism, Mutagenesis, Site-Directed, Coleoptera enzymology, Color, Luciferases chemistry, Luminescence, Luminescent Measurements
- Abstract
Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.
- Published
- 2016
- Full Text
- View/download PDF
41. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.
- Author
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Viviani VR, Simões A, Bevilaqua VR, Gabriel GV, Arnoldi FG, and Hirano T
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Animals, Catalytic Domain genetics, Coleoptera genetics, Fireflies enzymology, Fireflies genetics, Firefly Luciferin chemistry, Firefly Luciferin metabolism, Insect Proteins genetics, Insect Proteins metabolism, Kinetics, Luciferases genetics, Luciferases metabolism, Luciferases, Firefly chemistry, Luciferases, Firefly genetics, Luciferases, Firefly metabolism, Luminescent Measurements, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Coleoptera enzymology, Insect Proteins chemistry, Luciferases chemistry
- Abstract
Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also providing a base to accept excited oxyluciferin phenol proton, and a countercation to shield the negative charge of E311 and to stabilize excited oxyluciferin phenolate, blue-shifting emission spectra in most beetle luciferases.
- Published
- 2016
- Full Text
- View/download PDF
42. Organization and comparative analysis of the mitochondrial genomes of bioluminescent Elateroidea (Coleoptera: Polyphaga).
- Author
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Amaral DT, Mitani Y, Ohmiya Y, and Viviani VR
- Subjects
- Animals, Coleoptera classification, Genes, Mitochondrial, Luminescence, Phylogeny, RNA, Transfer genetics, Sequence Analysis, DNA, Coleoptera genetics, Genome, Mitochondrial
- Abstract
Mitochondrial genome organization in the Elateroidea superfamily (Coleoptera), which include the main families of bioluminescent beetles, has been poorly studied and lacking information about Phengodidae family. We sequenced the mitochondrial genomes of Neotropical Lampyridae (Bicellonycha lividipennis), Phengodidae (Brasilocerus sp.2 and Phrixothrix hirtus) and Elateridae (Pyrearinus termitilluminans, Hapsodrilus ignifer and Teslasena femoralis). All species had a typical insect mitochondrial genome except for the following: in the elaterid T. femoralis genome there is a non-coding region between NADH2 and tRNA-Trp; in the phengodids Brasilocerus sp.2 and P. hirtus genomes we did not find the tRNA-Ile and tRNA-Gln. The P. hirtus genome showed a ~1.6kb non-coding region, the rearrangement of tRNA-Tyr, a new tRNA-Leu copy, and several regions with higher AT contents. Phylogenetics analysis using Bayesian and ML models indicated that the Phengodidae+Rhagophthalmidae are closely related to Lampyridae family, and included Drilus flavescens (Drilidae) as an internal clade within Elateridae. This is the first report that compares the mitochondrial genomes organization of the three main families of bioluminescent Elateroidea, including the first Neotropical Lampyridae and Phengodidae. The losses of tRNAs, and translocation and duplication events found in Phengodidae mt genomes, mainly in P. hirtus, may indicate different evolutionary rates in these mitochondrial genomes. The mitophylogenomics analysis indicates the monophyly of the three bioluminescent families and a closer relationship between Lampyridae and Phengodidae/Rhagophthalmidae, in contrast with previous molecular analysis., (Copyright © 2016. Published by Elsevier B.V.)
- Published
- 2016
- Full Text
- View/download PDF
43. The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.
- Author
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Prado RA, Santos CR, Kato DI, Murakami MT, and Viviani VR
- Subjects
- Amino Acid Sequence, Animals, Coenzyme A Ligases chemistry, Coenzyme A Ligases metabolism, Coleoptera enzymology, Coleoptera metabolism, Crystallography, X-Ray, Esterification, Insect Proteins metabolism, Luciferases metabolism, Models, Molecular, Oxygenases metabolism, Protein Conformation, Protein Domains, Coleoptera chemistry, Insect Proteins chemistry, Luciferases chemistry, Oxygenases chemistry
- Abstract
Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343).
- Published
- 2016
- Full Text
- View/download PDF
44. Preparation and Assay of Simple Light off Biosensor Based on Immobilized Bioluminescent Bacteria for General Toxicity Assays.
- Author
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Gabriel GV and Viviani VR
- Subjects
- Bacteria drug effects, Gene Expression, Genes, Reporter, Hydrogen-Ion Concentration, Luciferases, Firefly genetics, Luciferases, Firefly metabolism, Microbial Viability drug effects, Bacteria genetics, Bacteria metabolism, Biosensing Techniques, Light, Luminescent Measurements methods, Toxicity Tests methods
- Abstract
We describe a simple light off bioluminescent biosensor for general environmental toxicity assays based on immobilized bioluminescent bacteria engineered with beetle luciferases.
- Published
- 2016
- Full Text
- View/download PDF
45. A transcriptional and proteomic survey of Arachnocampa luminosa (Diptera: Keroplatidae) lanterns gives insights into the origin of bioluminescence from the Malpighian tubules in Diptera.
- Author
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Silva JR, Amaral DT, Hastings JW, Wilson T, and Viviani VR
- Subjects
- Animals, Luciferases metabolism, Proteomics, Transcription, Genetic, Diptera chemistry, Insect Proteins genetics, Luciferases genetics, Luminescence, Malpighian Tubules chemistry, Proteome analysis, Transcriptome
- Abstract
Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera., (Copyright © 2015 John Wiley & Sons, Ltd.)
- Published
- 2015
- Full Text
- View/download PDF
46. Correction: A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties.
- Author
-
Viviani VR, Amaral D, Prado R, and Arnoldi FG
- Abstract
Correction for 'A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties' by Vadim R. Viviani et al., Photochem. Photobiol. Sci., 2011, 10, 1879-1886.
- Published
- 2015
- Full Text
- View/download PDF
47. First transcriptional survey of the Malpighian tubules of giant mealworm, Zophobas morio (Coleoptera: Tenebrionidae).
- Author
-
Silva JR, Prado RA, Amaral DT, and Viviani VR
- Subjects
- Animals, Insect Proteins genetics, Insect Proteins metabolism, Larva genetics, Mitochondria metabolism, Coleoptera genetics, Malpighian Tubules metabolism, Transcription, Genetic
- Abstract
The Malpighian tubules play a key role in insect osmoregulation. Although a transcriptional analysis has been done for the Malpighian tubules in Drosophila melanogaster (Diptera), no functional genomics analysis has yet been carried out for any Coleoptera species. Recently, we constructed a cDNA library from Malpighian tubules of larval Zophobas morio, a close relative of Tribolium castaneum, and cloned the cDNA for an AMP/CoA-ligase with luciferase-like enzyme properties. Using this cDNA library, we randomly isolated, partially sequenced and analyzed ca. 540 clones, obtaining the first transcriptional profile of the most representative expressed genes, and associated them with their possible biological functions. A high percentage of mitochondrial genes was found, which is consistent with the high metabolic activity required by this organ during the formation of primary urine. Common transcripts included those for enzymes involved in osmoregulation, such as solute transporters and ATPases, and in detoxification and excretion, such as cytochrome P450, glutathione S-transferase, alcohol dehydrogenase. The presence of AMP/CoA-ligases, which activate exogenous carboxylic acids such as firefly D-luciferin suggests their participation in important new xenobiotic excretion/detoxification roles in Malpighian tubule physiology.
- Published
- 2015
- Full Text
- View/download PDF
48. Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location.
- Author
-
Gabriel GV and Viviani VR
- Subjects
- Adenosine Triphosphate metabolism, Animals, Escherichia coli metabolism, Fireflies, Gene Expression, Hydrogen-Ion Concentration, Luminescence, Luminescent Measurements, Metals, Heavy metabolism, Plasmids genetics, Temperature, Transformation, Bacterial, Genes, Reporter, Intracellular Space metabolism, Luciferases, Firefly genetics, Luciferases, Firefly metabolism
- Abstract
Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes.
- Published
- 2014
- Full Text
- View/download PDF
49. Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.
- Author
-
Viviani VR, Neves DR, Amaral DT, Prado RA, Matsuhashi T, and Hirano T
- Subjects
- Animals, Binding Sites, Computational Biology, Firefly Luciferin chemistry, Luciferases chemistry, Molecular Structure, Protein Binding, Coleoptera enzymology, Color, Firefly Luciferin analogs & derivatives, Firefly Luciferin metabolism, Luciferases metabolism, Luminescent Agents chemistry, Phenols chemistry
- Abstract
Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar environment weakens such interaction promoting red shifts. In pH-sensitive luciferases, a pH-mediated switch from a closed hydrophobic conformation to a more open polar conformation promotes the typical red shift.
- Published
- 2014
- Full Text
- View/download PDF
50. Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.
- Author
-
Amaral DT, Arnoldi FG, Rosa SP, and Viviani VR
- Subjects
- Animals, Brazil, DNA, Ribosomal genetics, Fireflies chemistry, Insect Proteins genetics, Luciferases genetics, Luminescence, Molecular Sequence Data, Fireflies classification, Fireflies genetics, Phylogeny
- Abstract
Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae., (Copyright © 2013 John Wiley & Sons, Ltd.)
- Published
- 2014
- Full Text
- View/download PDF
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