Selective inhibition of Zophobas morio (Coleoptera: Tenebrionidae) luciferase-like enzyme luminescence by diclofenac and potential suitability for light-off biosensing.

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.

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.

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.

Nanobiosensors exploiting specific interactions between an enzyme and herbicides in atomic force spectroscopy.

The development of sensitive methodologies for detecting agrochemicals has become important in recent years due to the increasingly indiscriminate use of these substances. In this context, nanosensors based on atomic force microscopy (AFM) tips are useful because they provide higher sensitivity with operation at the nanometer scale. In this paper we exploit specific interactions between AFM tips functionalized with the enzyme acetolactate synthase (ALS) to detect the ALS-inhibitor herbicides metsulfuron-methyl and imazaquin. Using atomic force spectroscopy (AFS) we could measure the adhesion force between tip and substrate, which was considerably higher when the ALS-functionalized tip (nanobiosensor) was employed. The increase was approximately 250% and 160% for metsulfuron-methyl and imazaquin, respectively, in comparison to unfunctionalized probes. We estimated the specific enzyme-herbicide force by assuming that the measured force comprises an adhesion force according to the Johnson-Kendall-Roberts (JKR) model, the capillary force and the specific force. We show that the specific, biorecognition force plays a crucial role in the higher sensitivity of the nanobiosensor, thus opening the way for the design of similarly engineered tips for detecting herbicides and other analytes.

Nanobiosensors based on chemically modified AFM probes: a useful tool for metsulfuron-methyl detection.

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.

Luciferase from Fulgeochlizus bruchi (Coleoptera:Elateridae), a Brazilian click-beetle with a single abdominal lantern: molecular evolution, biological function and comparison with other click-beetle luciferases.

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.

The origin of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase): luciferin stereoselectivity as a switch for the oxygenase activity.

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.