Larval Tenebrio molitor (Coleoptera: Tenebrionidae) Fat Body Extracts Catalyze Firefly D-Luciferin- and ATP-Dependent Chemiluminescence: A Luciferase-like Enzyme*

Ultraweak light emission was detected upon injection of firefly luciferin into live Tenebrio larvae. A chemilumi-nescent enzymatic activity dependent on molecular oxygen, D-luciferin and MgATP was then isolated from larval fat body extracts by precipitation with 70% ammonium sulfate. D-Luciferin and ATP can be replaced by luciferyl-adenylate. Pyrophosphate is a main product from the chemiluminescent reaction. The in vitro chemiluminescence intensity was not affected by peroxidase inhibitors such as N₃^?_- (0.5 mM) and CN^? (1 mM), attesting to its nonperoxidatic nature but was strongly inhibited by AMP (1 mM), luciferin 6′-ethyl ether (1 mM) and sodium pyrophosphate (2 mM), well-known firefly lucifer-ase inhibitors. Some physical-chemical properties of this enzymatic activity were similar to those of firefly lucif-erase (K_MATP = 195 μM; K_0.5 luciferin - 0.8 mM; optimum pH 8.5; δ_max= 610 nm at pH 8.5; firefly lucifer-ase: δ_max= 565 nm at pH 8.0 and 619 mm at pH 6.0), but the chemiluminescence was not affected by addition of polyclonal antibodies raised against Photinus pyralis luciferase. These data suggest that this chemiluminescence results from a ligase with luciferase activity.     

Luciferin‐Regenerating Enzyme Crystal Structure Is Solved but its Function Is Still Unclear

Contribution of luciferin‐regenerating enzyme (LRE) for in vitro recycling of D‐luciferin has been reported. According to crystal structure of LRE, it is a beta‐propeller protein which is a type of all β‐protein architecture. In this overview, reinvestigation of the luciferase‐based LRE assays and its function is reported. Until now, sequence of LRE genes from four different species of firefly has been reported. In spite of previous reports, T‐LRE (from Lampyris turkestanicus) was cloned and expressed in Escherichia coli as well as Pichia pastoris in a nonsoluble form as inclusion body. According to recent investigations, bioluminescent signal of soluble T‐LRE–luciferase‐coupled assay increased and then reached an equilibrium state in the presence of D‐cysteine. In addition, the results revealed that both D‐ and L‐cysteine in the absence of T‐LRE caused a significant increase in bioluminescence intensity of luciferase over a long time. Based on activity measurements and spectroscopic results, D‐cysteine increased the activity of luciferase due to its redox potential and induction of conformational changes in structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE (at least T‐LRE) on luciferase activity, most of the increase in luciferase activity is caused by direct effect of D‐cysteine on structure and activity of firefly luciferase. Moreover, bioinformatics analysis cannot support the presence of LRE in peroxisome of photocytes in firefly lanterns.     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Unlike the enchanting yellow‐green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H‐bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination.     

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.     

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.     

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

The sensitive BRET system for the homogeneous immunoassay of a low‐molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)—the “red” mutant with λ_max.em = 590 nm (RedLuc) and the “green” mutant with λ_max.em = 550 nm (GreenLuc)—were tested as the donors. The water‐soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase–progesterone (Luc–Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF–Ab) were developed. Both conjugates retained their functional properties, had high antigen–antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL^?1.     

Luciferin‐Regenerating Enzyme Mediates Firefly Luciferase Activation Through Direct Effects of D‐Cysteine on Luciferase Structure and Activity

Luciferin‐regenerating enzyme (LRE) contributes to in vitro recycling of D‐luciferin. In this study, reinvestigation of the luciferase‐based LRE assay is reported. Here, using quick change site‐directed mutagenesis seven T‐LRE (Lampyris turkestanicusLRE) mutants were constructed and the most functional mutant of T‐LRE (T⁶⁹R) was selected for this research and the effects of D‐ and L‐cysteine on T⁶⁹R T‐LRE‐luciferase‐coupled assay are examined. Our results demonstrate that bioluminescent signal of T⁶⁹R T‐LRE‐luciferase‐coupled assay increases and then reach equilibrium state in the presence of 5 mm D‐cysteine. In addition, results reveal that 5 mm D‐ and L‐cysteine in the absence of T⁶⁹R T‐LRE cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far‐UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D‐cysteine increases the activity of luciferase due to weak redox potential, antiaggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time‐course originate from the direct effects of D‐cysteine on structure and activity of firefly luciferase.     

Functional and Photochemical Characterization of a Light‐Driven Proton Pump from the Gammaproteobacterium Pantoea vagans

Photoactive retinal proteins are widely distributed throughout the domains of the microbial world (i.e., bacteria, archaea, and eukarya). Here we describe three retinal proteins belonging to a phylogenetic clade with a unique DTG motif. Light‐induced decrease in the environmental pH and its inhibition by carbonyl cyanide m‐chlorophenylhydrazone revealed that these retinal proteins function as light‐driven outward electrogenic proton pumps. We further characterized one of these proteins, Pantoea vagans rhodopsin (PvR), spectroscopically. Visible spectroscopy and high‐performance liquid chromatography revealed that PvR has an absorption maximum at 538 nm with the retinal chromophore predominantly in the all‐trans form (>90%) under both dark and light conditions. We estimated the pK_a values of the protonated Schiff base of the retinal chromophore and its counterion as approximately 13.5 and 2.1, respectively, by using pH titration experiments, and the photochemical reaction cycle of PvR was measured by time‐resolved flash‐photolysis in the millisecond timeframe. We observed a blue‐shifted and a red‐shifted intermediate, which we assigned as M‐like and O‐like intermediates, respectively. Decay of the M‐like intermediate was highly sensitive to environmental pH, suggesting that proton uptake is coupled to decay of the M‐like intermediate. From these results, we propose a putative model for the photoreaction of PvR.     

Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Five new firefly luciferin ( 1 ) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains ( 2 – 4 ) and heterocyclic rings derived from amino acids ( 5 and 6 ) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin ( 6 ), possessing a pyrroline‐substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP‐carboluciferin (AMP=adenosine monophosphate) and AMP‐firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.     

Efficient Synthesis of 3‐Phenylnaphtho[2,3‐b]furan‐4,9‐diones in Water and Their Fluorimetric Study in Solutions

A simple and efficient protocol has been developed for the synthesis of 3‐phenylnaphtho[2,3‐b]furan‐4,9‐diones by domino reaction of α‐bromonitroalkenes to 2‐hydroxynaphthalene‐1,4‐dione. With the optimal reaction conditions [NaOAc (120 mol%), water, 70°C, 7 h], the scope of the domino reaction was explored and the green approach provided the desired products in moderate to good yields at elevated temperature under aqueous‐mediated conditions. A mechanistic rationalization for this reaction is also provided. The absorption characteristics of the compounds were examined by UV‐Vis spectra and fluorescence spectroscopy. All compounds were fluorescent in solution emitting at blue light (432–433 nm), green light (512–536 nm), or yellow light (591 nm).     

Firefly Chemiluminescence and Bioluminescence: Efficient Generation of Excited States

Firefly luciferase catalyzes a light‐emitting reaction in which an excited‐state product is formed. Both experimental and theoretical methodologies are used to study this system, and the reactions catalyzed by luciferase are relatively well characterized. However, the mechanism by which an excited‐state product is formed is still unknown. This Minireview deals with the current understanding of firefly bioluminescence and chemiluminescence. Thermal decomposition of simple 1,2‐dioxetanes is also discussed, due to their role in formation of the excited‐state bioluminophore.     

Cloning and characterization of an active fragment of luciferase from a luminescent marine alga, Pyrocystis lunula

Two marine dinoflagellates, Lingulodinium polyedrum and Pyrocystis lunula, emit light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen. The characteristic properties of P. lunula luciferase have not been clarified, whereas L. polyedrum luciferase, which has three active domains, has been characterized. A cloned partial cDNA of the P. lunula luciferase encodes an active fragment corresponding to part of domain 2 and all of domain 3 of L. polyedrum luciferase. The homology of the amino acid sequence between the two luciferases in domain 3 is about 84.3%. A recombinant His-tagged luciferase fragment containing domain 3 (Mr = 46 kDa) catalyzed the light-emitting oxidation of luciferin (lambdamax = 474 nm). This protein was purified by a single affinity-chromatography procedure. The pH-activity profile and the bioluminescence spectrum of the recombinant enzyme having a third domain are almost identical to those of an extract from P. lunula cultured in vitro. The recombinant enzyme is active at pH 8.0, although the recombinant enzyme derived from the second domain of L. polyedrum luciferase is inactive at pH 8.0. Substitution of Glu-201 by histidine in the third domain of P. lunula luciferase showed a decrease of activity above pH 7.0, suggesting that histidine residues could be responsible for pH-sensitivity in dinoflagellate luciferase.     

Poly(9‐undecyl‐9‐methyl‐fluorene) and poly(9‐pentadecyl‐9‐methyl‐fluorene): Synthesis,solution structure,and effect of side chain asymmetry on aggregation behavior

We report on solution aggregates and backbone conformation of poly(9‐undecyl‐9‐methyl‐fluorene) (PF1‐11) and poly(9‐pentadecyl‐9‐methyl‐fluorene) (PF1‐15), having two different side chains compared with poly(9,9‐dihexylfluorene) (PF6) and poly(9,9‐dioctylfluorene) (PF8) with two identical side chains. In the poor solvent methylcyclohexane (MCH), X‐ray scattering indicates that PF1‐11 and PF1‐15 appear as three‐dimensional aggregates (5–10 nm wide and thick), forming ribbon‐like agglomerates (correlation lengths of 100 nm). PF6 and PF8 appear as two‐dimensional aggregates (>10 nm wide and 2–3 nm thick) involving ribbon‐like agglomerates (correlation lengths much greater than 100 nm). Upon heating, all aggregates undergo a gel–sol transition which occurs at lower temperatures for PF1‐11 and PF1‐15 (<60°C) than for PF6 and PF8 (>80°C). In the good solvent toluene, PF1‐11 and PF1‐15 form networks of cylindrical particles. The mesh size and the cylinder radius are smaller in 24°C toluene (60 nm, 0.5 nm) than in 60°C MCH (300 nm, 1–2 nm). Nuclear magnetic resonance spectra in toluene‐d₈ together with density functional theory calculations suggest higher torsion angles between polymer repeat units for PF6, PF8, and PF1‐11 (less planar conformation) and a gauche arrangement of the dihedral angles between the bridge carbon atom and the side chain methylene groups in PF1‐15. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019 , 57, 826–837     

Photoinduced Ratchet‐Like Rotational Motion of Branched Molecular Crystals

Photomechanical molecular crystals can undergo a variety of light‐induced motions, including expansion, bending, twisting, and jumping. The use of more complex crystal shapes may provide ways to turn these motions into useful work. To generate such shapes, pH‐driven reprecipitation has been used to grow branched microcrystals of the anthracene derivative 4‐fluoroanthracenecarboxylic acid. When these microcrystals are illuminated with light of λ=405 nm, an intermolecular [4+4] photodimerization reaction drives twisting and bending of the individual branches. These deformations drive a rotation of the overall crystal that can be repeated over multiple exposures to light. The magnitude and direction of this rotation vary because of differences in the crystal shape, but a typical branched crystal undergoes a 50° net rotation after 25 consecutive irradiations for 1 s. The ability of these crystals to undergo ratchet‐like rotation is attributed to their chiral shape.