RESUMO

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 (IC50 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.

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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.

RESUMO

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.

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The use of agrochemicals has increased considerably in recent years, and consequently, there has been increased exposure of ecosystems and human populations to these highly toxic compounds. The study and development of methodologies to detect these substances with greater sensitivity has become extremely relevant. This article describes, for the first time, the use of atomic force spectroscopy (AFS) in the detection of enzyme-inhibiting herbicides. A nanobiosensor based on an atomic force microscopy (AFM) tip functionalised with the acetolactate synthase (ALS) enzyme was developed and characterised. The herbicide metsulfuron-methyl, an ALS inhibitor, was successfully detected through the acquisition of force curves using this biosensor. The adhesion force values were considerably higher when the biosensor was used. An increase of ~250% was achieved relative to the adhesion force using an unfunctionalised AFM tip. This considerable increase was the result of a specific interaction between the enzyme and the herbicide, which was primarily responsible for the efficiency of the nanobiosensor. These results indicate that this methodology is promising for the detection of herbicides, pesticides, and other environmental contaminants.

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Bioluminescent click-beetles emit a wide range of bioluminescence colors (λ(Max) = 534-594 nm) from thoracic and abdominal lanterns, which are used for courtship. Only the luciferases from Pyrophorus and Pyrearinus species were cloned and sequenced. The Brazilian Fulgeochlizus bruchi click-beetle, which inhabits the Central-west Cerrado (Savannas), is noteworthy because, differently from other click-beetles, the adult stage displays only a functional abdominal lantern, which produces a bright green bioluminescence for sexual attraction purposes, and lacks functional thoracic lanterns. We cloned the cDNA for the abdominal lantern luciferase of this species. Notably, the primary sequence of this luciferase showed slightly higher identity with the green emitting dorsal lantern luciferases of the Pyrophorus genus instead of the abdominal lanterns luciferases. This luciferase displays a blue-shifted spectrum (λ(Max) = 540 nm), which is pH-insensitive from pH 7.5 to 9.5 and undergoes a slight red shift and broadening above this pH; the lowest K(M) for luciferin among studied click-beetle luciferases, and the highest optimum pH (9.0) ever reported for a beetle luciferase. At pH 9.0, the K(M) for luciferin increases, showing a decrease of affinity for this substrate, despite the higher activity. The slow luminescence decay rate of F. bruchi luciferase in vitro reaction could be an adaptation of this luciferase for the long and sustained in vivo luminescence display of the click-beetle during the courtship, and could be useful for in vivo intracellular imaging.

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Firefly luciferases usually produce bioluminescence in the yellow-green region, with colors in the green and yellow-orange extremes of the spectrum being less common. Several firefly luciferases have already been cloned and sequenced, and site-directed mutagenesis studies have already identified important regions and residues for bioluminescence colors. However the structural determinants and mechanisms of bioluminescence colors turned out to be elusive, mainly when comparing luciferases with a high degree of divergence. Thus comparison of more similar luciferases producing colors in the two extremes of the spectrum could be revealing. The South-American fauna of fireflies remains largely unstudied, with some unique taxa that are not found anywhere else in the world and that produce a wide range of bioluminescence colors. Among them, fireflies of the genus Amydetes are especially interesting because its taxonomical status as an independent subfamily or as a tribe is not yet solved, and because they usually produce a continuous bright blue-shifted bioluminescence. In this work we cloned the cDNA for the luciferase of the Atlantic rain forest Amydetes fanestratus firefly, which is found near Sorocaba municipality (São Paulo, Brazil). Despite showing a higher degree of identity with the South-American Cratomorphus, the European Lampyris and the Asiatic Pyrocoelia, phylogenetical analysis of the luciferase sequence support the inclusion of Amydetes as an independent subfamily. Amydetes luciferase displays one of the most blue-shifted emission spectra (λ(max) = 538 nm) among beetle luciferases, with lower pH-sensitivity and higher affinity for ATP when compared to other luciferases, making this luciferase attractive for sensitive ATP and reporter assays.

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Beetle luciferases evolved from AMP/CoA-ligases. However, it is unclear how the new luciferase activity evolved. In order to clarify this question, we compared the luminescence and catalytic properties of a recently cloned luciferase-like enzyme from Zophobas mealworm, an AMP/CoA-ligase displaying weak luminescence activity, with those of cloned luciferases from the three main families of luminescent beetles: Phrixthrix hirtus railroad worm; Pyrearinus termitilluminans click beetle and Photinus pyralis firefly. The catalytic constant of the mealworm enzyme was 2-4 orders of magnitude lower than that of beetle luciferases, but 3 orders of magnitude above the non-catalyzed chemiluminescence of luciferyl-adenylate in buffer. Studies with D- and L-luciferin and their adenylates show that the luminescence reaction of the luciferase-like enzyme and beetle luciferases are stereoselective for D-luciferin and its adenylate, and that the selectivity is determined mainly at the adenylation step. Modelling studies showed that the luciferin binding site cavity of this enzyme is smaller and more hydrophobic than that of beetle luciferases. Therefore Zophobas mealworm enzyme displays true luciferase activity, keeping the attributes of an ancient protoluciferase. These results suggest that stereoselectivity for D-luciferin may have been a key event for the origin of oxygenase/luciferase activity in AMP/CoA-ligases, and that efficient luciferase activity may have further evolved mainly by increasing the catalytic constant of the oxidative reaction and the quantum yield of bioluminescence.

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Adult males of Eidmanacris corumbatai Garcia have reduced tegmina without stridulatory apparatus. For this reason, they developed other means ofintra-specific communication. During courtship, the males use a combination of foreleg drumming and waving of the antennae, in addition to chemical signaling through pheromones. The females become receptive to copulation when the males expose their metanotal gland. This gland, located on the male metanotum, is also a source of substances on which females feed before receiving the spermatophore. During copulation, the female destroys the apex of the metanotal gland to gain access to the secretion released by this structure.

Assuntos

RESUMO

Adult males of Eidmanacris corumbatai Garcia have reduced tegmina without stridulatory apparatus. For this reason, they developed other means of intra-specific communication. During courtship, the males use a combination of foreleg drumming and waving of the antennae, in addition to chemical signaling through pheromones. The females become receptive to copulation when the males expose their metanotal gland. This gland, located on the male metanotum, is also a source of substances on which females feed before receiving the spermatophore. During copulation, the female destroys the apex of the metanotal gland to gain access to the secretion released by this structure.

Em Eidmanacris corumbatai Garcia, os machos adultos apresentam tegminas reduzidas e sem aparelho estridulatório e, portanto, tiveram que desenvolver outros modos de comunicação intra-especifica. Durante a corte, os machos usam uma combinação de batimentos com as pernas dianteiras e ondulações das antenas, além da sinalização química através de feromônios, uma vez que as fêmeas se tornam receptivas à cópula quando os machos expõem a glândula metanotal. Essa glândula, localizada no metanoto do macho, é também uma fonte de substâncias das quais a fêmea se alimenta antes de receber o espermatóforo. Durante a cópula, a fêmea destrói o ápice da glândula metanotal para ter acesso à secreção liberada por essa estrutura.

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The present study compares the metanotal gland through scanning electron microscopy in five species of Eidmanacris: E. corumbatai Garcia, 1998, E. alboannulata (Piza, 1960), E. dissimilis Desutter-Grandcolas, 1995, E. larvaeformis (Chopard, 1938) and Eidmanacris sp. The general external configuration of the gland was determined by the presence of two median projections with apical opening and a cluster of bristles just above these projections. Although there is a general pattern for this gland, each species has its own pattern, which can be defined mainly by the arrangement of the bristles and the position of the median projections. Our results suggest the taxonomical importance of these structures, which should be better analyzed when describing species of the genus Eidmanacris. In addition, while observing the reproductive behavior of these species, we concluded that the release of this gland secretion is important for the success of mating.

O presente trabalho compara a glândula metanotal de cinco espécies do gênero Eidmanacris por meio de microscopia eletrônica de varredura: E. corumbatai Garcia, 1998, E. alboannulata (Piza, 1960), E. dissimilis Desutter-Grandcolas, 1995, E. larvaeformis (Chopard, 1938) e Eidmanacris sp. A configuração externa da glândula foi determinada pela presença de duas projeções medianas que apresentam abertura em seu ápice e um grupo de cerdas abaixo destas projeções. Apesar de haver um padrão geral semelhante, a configuração da glândula é espécie-específica, que pode ser definida principalmente pela organização de cerdas e pela posição das projeções medianas. Nossos resultados sugerem a importância taxonômica desta estrutura, que deveria ser melhor analisada na descrição de espécies do gênero Eidmanacris. Pela observação do comportamento reprodutivo destas espécies, nós concluímos que a liberação da secreção desta glândula é importante para o sucesso reprodutivo.

RESUMO

The present study compares the metanotal gland through scanning electron microscopy in five species of Eidmanacris: E. corumbatai Garcia, 1998, E. alboannulata (Piza, 1960), E. dissimilis Desutter-Grandcolas, 1995, E. larvaeformis (Chopard, 1938) and Eidmanacris sp. The general external configuration of the gland was determined by the presence of two median projections with apical opening and a cluster of bristles just above these projections. Although there is a general pattern for this gland, each species has its own pattern, which can be defined mainly by the arrangement of the bristles and the position of the median projections. Our results suggest the taxonomical importance of these structures, which should be better analyzed when describing species of the genus Eidmanacris. In addition, while observing the reproductive behavior of these species, we concluded that the release of this gland secretion is important for the success of mating.

O presente trabalho compara a glândula metanotal de cinco espécies do gênero Eidmanacris por meio de microscopia eletrônica de varredura: E. corumbatai Garcia, 1998, E. alboannulata (Piza, 1960), E. dissimilis Desutter-Grandcolas, 1995, E. larvaeformis (Chopard, 1938) e Eidmanacris sp. A configuração externa da glândula foi determinada pela presença de duas projeções medianas que apresentam abertura em seu ápice e um grupo de cerdas abaixo destas projeções. Apesar de haver um padrão geral semelhante, a configuração da glândula é espécie-específica, que pode ser definida principalmente pela organização de cerdas e pela posição das projeções medianas. Nossos resultados sugerem a importância taxonômica desta estrutura, que deveria ser melhor analisada na descrição de espécies do gênero Eidmanacris. Pela observação do comportamento reprodutivo destas espécies, nós concluímos que a liberação da secreção desta glândula é importante para o sucesso reprodutivo.