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.     

Improvement of Thermostability and Activity of Firefly Luciferase Through [TMG][Ac] Ionic Liquid Mediator

Firefly luciferase catalyzes production of light from luciferin in the presence of Mg²⁺?CATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K _m for ATP.     

THE COLORS OF FIREFLY BIOLUMINESCENCE—II EXPERIMENTAL EVIDENCE FOR THE OPTIMIZATION MODEL*

Abstract— The shapes, the peak wavelengths and the close matching of bioluminescence colors to visual spectral sensitivities in North American firefly species are consistent with the predictions of a spectral optimization model for selection in evolution (Seliger et al., 1982). A screening pigment found by microspectrophotometry in the rhabomeres of Photinus pyralis has the absorbancc characteristics predicted by the model. The biologically effective adaptation, a dimensionless ratio proportional to the relative advantage of a species to detect bioluminescence during twilight. has been calculated from experimentally determined distributions of ambient spectral radiances, visual spectral sensitivities and bioluminescence emissions and is shown to correlate both with color of bioluminescence and with the timing of initiation of flashing activity. The colors of firefly bioluminescence are therefore species-specific adaptations to optimize the detection of bioluminescence in the different photic environments in which the species have evolved.     

Quantum yields and quantitative spectra of firefly bioluminescence with various bivalent metal ions

We measured quantitative spectra of firefly (Photinus pyralis) bioluminescence in the presence of Zn²⁺ and other bivalent metal ions to investigate the effects of these metal ions on luciferin‐luciferase reaction. We studied the dependence of the quantum yield and spectrum on quantity and kind of bivalent metal ions. Adding various amounts of Mg²⁺, Mn²⁺ and Ca²⁺ produced virtually no change in the quantum yields or the spectra of bioluminescence. In contrast, increasing amounts of ions such as Zn²⁺ and Cd²⁺ decreased quantum yields and changed the bioluminescence color from yellow‐green to red. Quantitative analysis showed that the sensitivities of the quantum yield and color to various metal ions were in the order of Hg²⁺>Zn²⁺, Cd²⁺>Ni²⁺, Co²⁺, Fe²⁺≫Mg²⁺, Mn²⁺, Ca²⁺. We propose that the changes in quantum yield and spectrum caused by the metal ions are due to their effect on luciferase that surrounds oxyluciferin during its radioactive decay. We also found that having more metal ions accelerated bioluminescence reactions. The sensitivity of the reaction rate had no correlation with those of the quantum yield and spectrum.     

Directed Improvement of Luciferin Regenerating Enzyme Binding Properties: Implication of Some Conserved Residues in Luciferin‐Binding Domain

Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of d ‐luciferin to produce persistent and longer light emission by luciferase. Luciferin binding domains I and II among LREs regarded as potential candidates for luciferin‐binding sites. In this study, for the first time, amino acids T69, G75 and K77 located at luciferin binding domain I of LRE from L. turkestanicus (T‐LRE) substituted by using site‐directed mutagenesis. Single mutant T⁶⁹R increased luciferase light output more than two‐fold over a longer time in comparison with a wild‐type and other mutants of T‐LRE. Nevertheless, double mutant (K⁷⁷E/T⁶⁹R) increased the amount of bioluminescent signal more than two‐fold over a short time. In addition, G⁷⁵E, K⁷⁷E and G⁷⁵E/T⁶⁹R mutants did not improve luciferin–luciferase in vitro bioluminescence. Based on our results, addition of K⁷⁷E/G⁷⁵E and K⁷⁷E/G⁷⁵E/T⁶⁹R mutants caused intermediate changes in bioluminescence from in vitro luciferin–luciferase reaction. These findings indicated that the amino acids in question are possible to be located within T‐LRE active site. It may also be suggested that substituted Arg69 (Arg218) plays an important role in luciferin binding and the existence of Gly75 as well as Lys77 is essential for T‐LRE which has already evolved to have different functions in nature.     

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.     

The stress-related production of the activePhotinus pyralis and luciolaMingrelica firefly luciferases inEscherichia coli

The kinetics ofPhotinus pyralis andLuciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible λP_r promoter inEscherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay.E. coli cells were cultivated at temperature schemes 28–42–21°C or 28–21°C, or at alkali pH shift. In the cases of thermoinduction and pH shift, the luciferase expressions have similar features. The 3-h thermoinduction (42°C) followed by the incubation at 21°C, for 10 h resulted in the maximal amount of the luciferase protein of 4–5% of the total cell proteins. The yield did not change further. The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21°C and reached the maximum after 50–60 h when the synthesized luciferase protein adopted the native-like conformation. At the same time, only 50% of the latter appeared to be catalytically active. An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content. Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.     

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.     

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.     

Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo bioluminescence imaging

The dimethylamino group of AkaLumine ((4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazolecarboxylic acid), a red-light-emitting firefly luciferin analogue, was replaced by cyclic amino groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl, and 4-morpholino) to give AkaLumine analogues exhibiting desirable bioluminescence with emission maxima in the red region (656–667 nm). In particular, a bioluminescence reaction of 1-pyrrolidinyl analogue with a recombinant Photinus pyralis luciferase showed a higher quantum yield than that with AkaLumine, giving an improved bioluminescence intensity. The 1-pyrrolidinyl analogue also showed the strongest luminescence in whole-body luciferase-expressing mice among the analogues, indicating that a quantum yield improvement of a luciferin analogue is effective to increase bioluminescence imaging intensity.     

Multi-function fibre-optic sensor for the bioluminescent flow determination of ATP or NADH

A multi-function biosensor for the determination of either ATP or NADH using a single bioluminescence-based fibre-optic probe is described. This was made possible by co-immobilizing the firefly luciferase from Photinus pyralis for ATP analysis with the bacterial/oxidoreductase system from Vibrio harveyi for NADH analysis, on the same preactivated polyamide membrane. Compatible analytical conditions with regard to the activity and stability of each bioluminescent system were selected, enabling them to attain their highest performances. It was possible to perform continuous-flow measurements of ATP and NADH over a wide (log-log) linear calibration range with a relative standard deviation of 4.0–4.5% and detection limits of 0.25 pmol ATP and 5 pmol NADH.     

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.     

Yellow-green and red firefly bioluminescence from 5,5-dimethyloxyluciferin

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) similar 530 nm) to red (lambda(max) similar 635 nm). The original mechanism of bioluminescence color determination advanced by White and co-workers was based on the concept that the keto and enol tautomers of the emitter oxyluciferin produce red and green light, respectively. Alternatively, McCapra proposed that color variation is associated with conformations of the keto form of excited-state oxyluciferin. We have prepared the adenylate of D-5,5-dimethylluciferin and shown that it is transformed into the putative emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the green-emitting click beetle. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, may be taken as the first experimental support for McCapra's mechanism of firefly bioluminescence color or any other proposal that requires only a single keto form of oxyluciferin.     

Synthesis of Macrocyclic Insect‐Derived Alkaloids

Macrocyclic lactonic alkaloids found in the pupal secretions of two species of a coccinellid beetle (genus Epilachna) were prepared in enantiomerically pure form via an efficient synthetic route using enantiomerically pure α‐amino acids as chiral‐pool starting materials. Macrocycles with rings containing up to 98 atoms were synthesized in good yield using Mukaiyama's macrolactonization conditions.     

Exploring Bioluminescence Function of the Ca2+‐regulated Photoproteins with Site‐directed Mutagenesis

Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca²⁺‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.