Bioluminescence Reaction Research Articles

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

Evidence for the generation of myristylated FMN by bacterial luciferase.

The genes responsible for the light production in bioluminescent bacteria are present as an operon, luxCDABEG. Many strains of Photobacteria carry an additional gene, termed luxF. X-ray crystallographic analysis of LuxF revealed the presence of four molecules of a flavin derivative, i.e. 6-(3'-(R)-myristyl) flavin adenine mononucleotide (myrFMN) non-covalently bound to the homodimer. In the present study, we exploited the binding of myrFMN to recombinant apo-LuxF to explore the occurrence of myrFMN in various bioluminescent bacteria. MyrFMN was detected in all bacterial strains tested including Vibrio and Aliivibrio indicating that it is more widely occurring in bioluminescent bacteria than previously assumed. We also show that apo-LuxF captures myrFMN and thereby relieves the inhibitory effect on luciferase activity. Thus our results provide support for the hypothesis that LuxF acts as a scavenger of myrFMN in bioluminescent bacteria. However, the source of myrFMN remained obscure. To address this issue, we established a cofactor regeneration enzyme-catalyzed cascade reaction that supports luciferase activity in vitro for up to 3 days. This approach enabled us to unambiguously demonstrate that myrFMN is generated in the bacterial bioluminescent reaction. Based on this finding we postulate a reaction mechanism for myrFMN generation that is based on the luciferase reaction.

Variability of fluorescence spectra of coelenteramide-containing proteins as a basis for toxicity monitoring

Nowadays, physicochemical approach to understanding toxic effects remains underdeveloped. A proper development of such mode would be concerned with simplest bioassay systems. Coelenteramide-Containing Fluorescent Proteins (CLM-CFPs) can serve as proper tools for study primary physicochemical processes in organisms under external exposures. CLM-CFPs are products of bioluminescent reactions of marine coelenterates. As opposed to Green Fluorescent Proteins, the CLM-CFPs are not widely applied in biomedical research, and their potential as colored biomarkers is undervalued now. Coelenteramide, fluorophore of CLM-CFPs, is a photochemically active molecule; it acts as a proton donor in its electron-excited states, generating several forms of different fluorescent state energy and, hence, different fluorescence color, from violet to green. Contributions of the forms to the visible fluorescence depend on the coelenteramide microenvironment in proteins. Hence, CLM-CFPs can serve as fluorescence biomarkers with color differentiation to monitor results of destructive biomolecule exposures. The paper reviews experimental and theoretical studies of spectral-luminescent and photochemical properties of CLM-CFPs, as well as their variation under different exposures – chemicals, temperature, and ionizing radiation. Application of CLM-CFPs as toxicity bioassays of a new type is justified.

Taking Electrons out of Bioelectronics: From Bioprotonic Transistors to Ion Channels.

From cell‐to‐cell communication to metabolic reactions, ions and protons (H+) play a central role in many biological processes. Examples of H+ in action include oxidative phosphorylation, acid sensitive ion channels, and pH dependent enzymatic reactions. To monitor and control biological reactions in biology and medicine, it is desirable to have electronic devices with ionic and protonic currents. Here, we summarize our latest efforts on bioprotonic devices that monitor and control a current of H+ in physiological conditions, and discuss future potential applications. Specifically, we describe the integration of these devices with enzymatic logic gates, bioluminescent reactions, and ion channels.

Development of Highly Sensitive Analytical Methods for Biologically Relevant Materials and Their Pharmaceutical Applications

One important aspect of analytical chemistry research in the pharmaceutical sciences is the development of diagnostic and therapeutic analyses for disease, and the development of analytical methods for elucidating the causes of disease. I have been focusing on developing a highly sensitive method for measuring trace amounts of specific components in biological samples. This research can be roughly divided into three approaches: the use of immunoassays and DNA hybridization as methods utilizing specific affinities, the use of capillary electrophoresis as a highly sensitive and rapid separation method, and the use of chemiluminescence and bioluminescence reactions. The components being measured are compounds such as hormones, tumor markers, drugs, reactive oxygen species and genes in biological samples for the purpose of developing therapies for the prevention and treatment of diseases.

Mechanism of activated chemiluminescence of cyclic peroxides: 1,2-dioxetanes and 1,2-dioxetanones.

Almost all chemiluminescent and bioluminescent reactions involve cyclic peroxides. The structure of the peroxide and reaction conditions determine the quantum efficiency of light emission. Oxidizable fluorophores, the so-called activators, react with 1,2-dioxetanones promoting the former to their first singlet excited state. This transformation is inefficient and does not occur with 1,2-dioxetanes; however, they have been used as models for the efficient firefly bioluminescence. In this work, we use the SA-CASSCF/CASPT2 method to investigate the activated chemiexcitation of the parent 1,2-dioxetane and 1,2-dioxetanone. Our findings suggest that ground state decomposition of the peroxide competes efficiently with the chemiexcitation pathway, in agreement with the available experimental data. The formation of non-emissive triplet excited species is proposed to explain the low emission efficiency of the activated decomposition of 1,2-dioxetanone. Chemiexcitation is rationalized considering a peroxide/activator supermolecule undergoing an electron-transfer reaction followed by internal conversion.

Luciferase-based bioassay for rapid pollutants detection and classification by means of multilayer artificial neural networks

Biosensors for rapid environmental pollution detection can be designed with biomodule based on the bacterial bioluminescent system. Usually this method returns total value of toxicity and does not allow to distinguish pollutants types. Herein we demonstrate the classification of pollutants by the kinetic analysis utilizing artificial neural networks with multilayer perceptron architecture. The kinetics of light emission of NAD(P)H:FMN-oxidoreductase-luciferase bioluminescent reaction was measured for clean water and in the presence of three environment pollutants (1,4-benzoquinone, copper sulfate and 1,3-dihydroxybenzene) separately with various concentrations. The efficiency of using multilayer perceptron with sigmoid activation function for processing of kinetics of light emission was estimated. It was shown that multilayer perceptrons allowing to distinguish pollutant class and concentration after sufficient training. The architecture consisted of 61 inputs neurons, 3 hidden layers and 3 output neurons was found optimum in sense of learning time for classification of three pollutants. Usage of simplest activation function sigmoid and backpropagation method for multilayer perceptron teaching providing the results been useful for smart signal processing in computational modules of biosensors.

A Tale Of Two Luciferins: Fungal and Earthworm New Bioluminescent Systems.

Bioluminescence, the ability of a living organism to produce light through a chemical reaction, is one of Nature's most amazing phenomena widely spread among marine and terrestrial species. There are various different mechanisms underlying the emission of "cold light", but all involve a small molecule, luciferin, that provides energy for light-generation upon oxidation, and a protein, luciferase, that catalyzes the reaction. Different species often use different proteins and substrates in the process, which suggests that the ability to produce light evolved independently several times throughout evolution. Currently, it is estimated that there are more than 30 different mechanisms of bioluminescence. Even though the chemical foundation underlying the bioluminescence phenomenon is by now generally understood, only a handful of luciferins have been isolated and characterized. Today, the known bioluminescence reactions are used as indispensable analytical tools in various fields of science and technology. A pressing need for new bioluminescent analytical techniques with a wider range of practical applications stimulates the search and chemical studies of new bioluminescent systems. In the past few years two such systems were unraveled: those of the earthworms Fridericia heliota and the higher fungi. The luciferins of these two systems do not share structural similarity with the previously known ones. This Account will survey structure elucidation of the novel luciferins and identification of their mechanisms of action. Fridericia luciferin is a key component of a novel ATP-dependent bioluminescence system. Structural studies were performed on 0.005 mg of natural substance and revealed its unusual extensively modified peptidic nature. Elucidation of Fridericia oxyluciferin revealed that oxidative decarboxylation of a lysine fragment of luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as a light emitter. Along with luciferin, a number of its natural analogs were found in the extracts of worm biomass. They occurred to be highly unusual modified peptides comprising a set of amino acids, including threonine, aminobutyric acid, homoarginine, unsymmetrical N,N-dimethylarginine and extensively modified tyrosine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and raise novel questions concerning their biosynthetic origin. Also in this Account we discuss identification of the luciferin of higher fungi 3-hydroxyhispidin which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Multicolor Bioluminescence Obtained Using Firefly Luciferin.

Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study

Coelenteramide-containing fluorescent proteins are products of bioluminescent reactions of marine coelenterates. They are called ‘discharged photoproteins’. Their light-induced fluorescence spectra are variable, depending considerably on external conditions. Current work studies a dependence of light-induced fluorescence spectra of discharged photoproteins obelin, aequorin, and clytin on excitation energy. It was demonstrated that photoexcitation to the upper electron-excited states (260–300nm) of the discharged photoproteins initiates a fluorescence peak in the near UV region, in addition to the blue-green emission. To characterize the UV fluorescence, the light-induced fluorescence spectra of coelenteramide (CLM), fluorophore of the discharged photoproteins, were studied in methanol solution. Similar to photoproteins, the CLM spectra depended on photoexcitation energy; the additional peak (330nm) in the near UV region was observed in CLM fluorescence at higher excitation energy (260–300nm). Quantum chemical calculations by time depending method with B3LYP/cc-pVDZ showed that the conjugated pyrazine-phenolic fragment and benzene moiety of CLM molecule are responsible for the additional UV fluorescence peak. Quantum yields of CLM fluorescence in methanol were 0.028±0.005 at 270–340nm photoexcitation. A conclusion was made that the UV emission of CLM might contribute to the UV fluorescence of the discharged photoproteins. The study develops knowledge on internal energy transfer in biological structures – complexes of proteins with low-weight aromatic molecules.

Structural distinctions of fast and slow bacterial luciferases revealed by phylogenetic analysis

Bacterial luciferases are heterodimeric enzymes that catalyze a chemical reaction, so called bioluminescence, which causes light emission in bacteria. Bioluminescence is vastly used as a reporter system in research tools and commercial developments. However, the details of the mechanisms that stabilize and transform the reaction intermediates as well as differences in the enzymatic kinetics amongst different bacterial luciferases remain to be elucidated. Amino acid sequences alignments for 21 bacterial luciferases (both α- and β-subunits) were analyzed. For α-subunit, containing the enzyme active center, 48 polymorphic amino acid positions were identified. According to them, the sequences fell into two distinct groups known as slow and fast based on the decay rate of the bioluminescence reaction. The differences in the enzyme active site induced by structural polymorphism are analyzed. Three-dimensional models of Photobacterium leiognathi luciferase and Vibrio harveyi luciferase (with reconstructed mobile loop) are freely available at PMDB database: PM0080525 and PM0080526, respectively. adeeva@sfu-kras.ruSupplementary information: Supplementary data are available at Bioinformatics online.

Mass spectrometry analysis and transcriptome sequencing reveal glowing squid crystal proteins are in the same superfamily as firefly luciferase.

The Japanese firefly squid Hotaru-ika (Watasenia scintillans) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1–3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1–3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates.

Evaluation of Oxidative Metabolism in Leukocytes during Phagocytosis of Escherichia coli Carrying Genetic Constructs soxS::lux or katG::lux.

We studied ROS generation by human peripheral blood monocytes and granulocytes during phagocytosis of Escherichia coli soxS::lux or katG::lux responding by luminescence (bioluminescence) to the development of oxidative stress. Initially high sensitivity of the bioluminescent reaction of E. coli katG::lux strain to the effects of model ROS (KO2 and H2O2) and pronounced induction of luminescence upon contact with granulocytes, whereas E. coli soxS::lux demonstrated less pronounced reaction to chemical oxidants and bioluminescence was observed primarily upon contact with monocytes. A correlation was found between quantitative characteristics of E. coli katG::lux bioluminescence and luminol-dependent chemiluminescence of leukocytes in some patients, but no dependence of this kind was noted for E. coli soxS::lux. The results can provide experimental substantiation of a new approach for evaluation of ROS production by leukocytes during phagocytosis and choosing the optimal object for these studies.

One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine.

Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine.

Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22% of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a general purpose model to understand, anticipate, or predict the dysfunction of a biosensor using immobilized bioluminescent bioreporter in a matrix.

The Light Emitter of the 2‐Coumar­anone Chemiluminescence: Theoretical and Experimental Elucidation of a Possible Model for Bioluminescent Systems

AbstractThe bioluminescence reaction of many biological subspecies, most notably fireflies, forms dioxetanone derivatives as high‐energy intermediates. The thermal instability of dioxetanones complicates understanding of the transition from the ground state to the first excited state that leads to light emission. Herein, we report the reaction mechanism of 2‐coumaranones, synthetically accessible strongly chemiluminescent materials that mimic the bioluminescence reaction. The pathways of chemiexcitation and photorelaxation are clarified on the basis of synthetic evidence and spectrochemical as well as computational mechanistic analysis.

Real-time monitoring of cariogenic bacteria via bioluminescent imaging: A biodontic hypothesis

Introduction: Dental caries (tooth decay) remains one of the most common chronic infectious disease in the world. Disclosure of camouflaged cariogenic bacteria will be a great motivation for better oral hygiene. The Hypothesis: At present, lux transposon cassette, Tn4001 luxABCDE Kmr, is available that could be used for stable bioluminescent transformation of a wide range of gram-positive bacteria, e.g. Streptococcus mutans and Lactobacillus. After this step, sensitive charge-coupled device (CCD) camera could be used to detect the low levels of light emitted from bioluminescent cariogenic bacteria. Living imaging software would be used for analysis and three-dimensional (3D) reconstruction of images. Evaluation of the Hypothesis: Entrance of transgenic organisms into the oral cavity should be done with great caution. Ethical consideration is necessary and primary animal studies are required. The main limitation of this technique will be oxygen. As mentioned previously, bioluminescent reactions need oxygen. Hence, bioluminescent imaging cannot be used for anaerobic bacteria, e.g., Streptococcus sobrinus.

Quantum Yield Determination Based on Photon Number Measurement, Protocols for Firefly Bioluminescence Reactions.

Quantum yield (QY), which is defined as the probability of photon production by a single bio/chemiluminescence reaction, is an important factor to characterize luminescence light intensity emitted diffusively from the reaction solution mixture. Here, methods to measure number of photons to determine QY according to the techniques of national radiometry standards are described. As an example, experiments using firefly bioluminescence reactions are introduced.

DNA Triplexes That Bind Several Cofactor Molecules.

Invited for the cover of this issue are Sven Vollmer and Clemens Richert of the University of Stuttgart. The cover image hints at the analogy between a honey comb, as a macroscopic storage device, and DNA triplexes with designed binding sites, as molecular storage motifs that can release ATP to fuel a bioluminescence reaction. Read the full text of the article at 10.1002/chem.201503220.

Immobilization of Firefly Luciferase on PVA-co-PE Nanofibers Membrane as Biosensor for Bioluminescent Detection of ATP.

The bioluminescent reaction catalyzed by firefly luciferase has become widely established as an outstanding analytical system for assay of adenosine triphosphate (ATP). When in solution, the luciferase is unstable and cannot be reused. The problem can be partially solved by immobilizing the luciferase on solid substrates. The poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibers membrane has abundant active hydroxyl groups on the surface. The PVA-co-PE nanofibers membrane was first activated by cyanuric chloride with triazinyl group. Then the activated PVA-co-PE nanofibers membrane was subsequently reacted with 1,3-propanediamine and biotin. The firefly luciferase was immobilized onto the surface of 1,3-propanediamine- and biotin-functionalized membranes. The surface chemical structure and morphologies of nanofibers membranes were characterized by FTIR-ATR spectra and SEM. The hydrophilicity of membranes was tested by water contact angle measurements. The detection of fluorescence intensity displayed that the firefly-luciferase-immobilized PVA-co-PE nanofibers membranes indicated high catalytic activity and efficiency. Especially, the firefly-luciferase-immobilized nanofiber membrane which was functionalized by biotin can be a promising candidate as biosensor for bioluminescent detection of ATP because of its high detection sensitivity.