Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.     

Orthologous gene of beetle luciferase in non-luminous click beetle, Agrypnus binodulus (Elateridae), encodes a fatty acyl-CoA synthetase

A homologous gene of beetle luciferase, AbLL (Agrypnus binodulusluciferase-like gene) was isolated from a Japanese non-luminous click beetle, A. binodulus, and its gene product was characterized. The identity of amino acid sequence deduced from AbLL with the click beetle luciferase from the Jamaican luminous click beetle, Pyrophorus plagiophthalmus, is 55%, which is higher than that between click beetle luciferase and firefly luciferase (approximately 48%). Phylogenetic analysis indicated that AbLL places in a clade of beetle luciferases, suggesting that AbLL is an orthologous gene of beetle luciferase. The gene product of AbLL (AbLL) has medium- and long-chain fatty acyl-CoA synthetase activity, but not luciferase activity. The fatty acyl-CoA synthetic activity was slightly inhibited in the presence of beetle luciferin, suggesting that AbLL has poor affinity for beetle luciferin. By comparing the amino acid residues of the catalytic domains in beetle luciferases with AbLL, the key substitutions for the luminescence activity in beetle luciferase will be proposed.     

Interaction of dimethyl-and monomethyloxyluciferin with recombinant wild-type and mutant firefly luciferases

Dissociation constants (Ks) in the pH range 6.5-9.0 for complexes of luciferin, dimethyloxyluciferin (DMOL), and monomethylluciferin (MMOL) with recombinant wild-type and mutant (His433Tyr) luciferases from the Luciola mingrelica firefly were determined by fluorescent titration. The protonated effectors were bound by the wild-type and mutant luciferases better than the nonprotonated ones. The affinity of DMOL for the mutant luciferase was higher than for the wild-type luciferase at alkaline pH, whereas the affinity of MMOL was higher at all pH values studied. The fluorescence emission and excitation spectra of DMOL and MMOL in buffer solution (pH 7.8) were obtained in the absence and presence of luciferase. The fluorescence maxima of DMOL and MMOL complexes with luciferase were 20 and 100 nm, respectively, shifted to shorter wavelengths as compared to the values in buffer solution. This was explained by nonspecific and specific influence of the protein microenvironment on the fluorescence spectra of DMOL and its specific influence on the MMOL fluorescence spectra.     

Vision in click beetles (Coleoptera: Elateridae): pigments and spectral correspondence between visual sensitivity and species bioluminescence emission

Among lampyrids, intraspecific sexual communication is facilitated by spectral correspondence between visual sensitivity and bioluminescence emission from the single lantern in the tail. Could a similar strategy be utilized by the elaterids (click beetles), which have one ventral abdominal and two dorsal prothoracic lanterns? Spectral sensitivity [S(λ)] and bioluminescence were investigated in four Brazilian click beetle species Fulgeochlizus bruchii, Pyrearinus termitilluminans, Pyrophorus punctatissimus and P. divergens, representing three genera. In addition, in situ microspectrophotometric absorption spectra were obtained for visual and screening pigments in P. punctatissimus and P. divergens species. In all species, the electroretinographic S(λ) functions showed broad peaks in the green with a shoulder in the near-ultraviolet, suggesting the presence of short- and long-wavelength receptors in the compound eyes. The long-wavelength receptor in Pyrophorus species is mediated by a P540 rhodopsin in conjunction with a species-specific screening pigment. A correspondence was found between green to yellow bioluminescence emissions and its broad S(λ) maximum in each of the four species. It is hypothesized that in elaterids, bioluminescence of the abdominal lantern is an optical signal for intraspecifc sexual communication, while the signals from the prothoracic lanterns serve to warn predators and may also provide illumination in flight.     

中国萤火虫云南窗萤荧光素酶cDNA的克隆表达和序列分析

从一种来自中国日行性萤火虫(云南窗萤)发光器官mRNA中克隆、测序并表达了有功能的荧光素酶.云南窗萤荧光素酶的cDNA序列有1647个碱基,编码548个氨基酸残基.从推测得到的氨基酸序列的比对分析得出:云南窗萤的荧光素酶与来自Lampyris noctiluca,L.turkestanicus和Nyctophila cf.caucasica三种萤火虫的荧光素酶有97.8%的序列一致性.从推测得出的氨基酸序列进行系统发育分析,其结果表明:云南窗萤和Lampyris Nyctophila聚在一起,与同属的发光强夜行性的萤火虫不形成的单系.云南窗萤荧光素酶在大肠杆菌中表达的条带大约70kDa,并且在有荧光素存在时发出黄绿色荧光.对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点.云南窗萤和窗萤属的其他3种萤火虫的荧光素酶卡目比,有13个不同氨基酸位点,位于模拟分子结构的表面.对于这些多肽环、不刚氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异.     

Spectral correspondence between visual spectral sensitivity and bioluminescence emission spectra in the click beetle Pyrophorus punctatissimus (Coleoptera: Elateridae)

The presence of two spectral mechanisms, near-ultraviolet and green (lambda(max)=545nm), is strongly suggested by electroretinographic visual spectral sensitivity curves obtained under dark and red chromatic adaptation conditions in the compound eyes of the click beetle Pyrophorus punctatissimus. The bioluminescence emission of the dorsal prothoracic lanterns is deep green (lambda(max)=543nm) and that of the ventral abdominal lantern is lime green (lambda(max)=556nm) in colour in P. punctatissimus. A broad green visual receptor would detect both deep green and lime green bioluminescent optical signals.     

Introduction to beetle luciferases and their applications

All beetle luciferases have evolved from a common ancestor: they all use ATP, O₂, and a common luciferin as substrates. The most studied of these luciferases is that derived from the firefly Photinus pyralis, a beetle in the superfamily of Cantharoidea. The sensitivity with which the activity of this enzyme can be assayed has made it useful in the measurement of minute concentrations of ATP. With the cloning of the cDNA coding this luciferase, it has also found wide application in molecular biology as a reporter gene. We have recently cloned other cDNAs that code for luciferases from the bioluminescent click beetle, Pyrophorus plagiophthalamus, in the superfamily Elateroidea. These newly acquired luciferases are of at least four different types, distinguishable by their ability to emit different colours of bioluminescence ranging from green to orange. Unique properties of these luciferases, especially their emission of multiple colours, may make them additionally useful in applications.     

Immobilization of luciferase from the firefly Luciola mingrelica — Catalytic properties and stability of the immobilized enzyme

Firefly (Luciola mingrelica) luciferase [Photinus luciferin 4-monooxygenase (ATP-hydrolysing); Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing), EC 1.13.12.7] has been immobilized on albumin and polyacrylamide gel, on AH-, CH- and CNBr-Sepharose 4B as well as on Ultragel, Ultradex and cellophane film activated by cyanogen bromide. Only immobilization on cyanogen bromide-activated polysaccharide carriers resulted in highly active immobilized luciferase. Kinetic properties of immobilized luciferase hardly differed from those of the soluble enzyme. The inactivation rate constants of soluble and immobilized luciferase were measured at pH 5.5–9.0 and 25°C as well as at pH 7.8 and 20–40°C. The ΔH^≠ and ΔS^≠ values for inactivation of soluble and immobilized luciferases were obtained. A 1000-fold stabilization effect was noted for the luciferase immobilized on CNBr-Sepharose 4B at pH 7.5 and 25°C. A stabilization mechanism for the immobilized luciferase is discussed.     

Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata

Luciferases of Japanese and North American fireflies act on a common substrate (luciferin) but the resulting lights emitted are of different colors. As a step toward an understanding of the molecular mechanism of the luciferase reaction, a cDNA clone (pGLf1) was isolated from a cDNA library of lantern poly(A)+RNA of the Japanese firefly, Luciola cruciata ('Genji-botaru' in Japanese), using a cDNA of North American firefly luciferase. The isolated 2-kb cDNA sequence was able to direct the synthesis of active luciferase in Escherichia coli under the control of the lac promoter. The primary structure of Genji firefly luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids (aa) with an Mr of 60,024. Homology between the amino acid sequences of the Genji and North American firefly luciferases was 67%, but a number of amino acid changes were found in the first 200 aa from the N terminus.     

Dinoflagellate bioluminescence: a comparative study of invitro components.

In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunual, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross-react in all combinations. All of these species possess high-molecular weight luciferases (200,000-400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW approximately 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.     

THE CIRCADIAN RHYTHM OF BIOLUMINESCENCE IN PYROCYSTIS IS NOT DUE TO DIFFERENCES IN THE AMOUNT OF LUCIFERASE: A COMPARATIVE STUDY OF THREE BIOLUMINESCENT MARINE DINOFLAGELLATES

The biochemistry and circadian regulation of luminescence in two Pyrocystis species, P. lunula Hulburt and P. noctiluca Murray et Haeckel, were compared with a well-studied species, Gonyaulax polyedra Stein. All exhibit circadian rhythms and all have similar luciferins and luciferases. However, the Pyrocystis species lack a second protein involved in the reaction in Gonyaulax , the luciferin (substrate) binding protein, which sequesters the luciferin at the cytoplasmic pH and releases it upon acidification, thus controlling the characteristic flashing, which is similar in the three species. More striking is the difference in the circadian regulation of luminescence, which in Gonyaulax involves the daily synthesis and destruction of the two proteins, along with the luminous organelles (scintillons) from which light is emitted, and which are present in all species. In the Pyrocystis species, the amount of luciferase is the same in extracts made during the day and night phases; its circadian regulation in vivo may be attributed to a change in its localization from day to night phase.     

中国萤火虫云南窗萤荧光素酶cDNA的克隆表达和序列分析(英文）

从一种来自中国日行性萤火虫（云南窗萤）发光器官mRNA中克隆、测序并表达了有功能的荧光素酶。云南窗萤荧光素酶的cDNA序列有1 647个碱基,编码548个氨基酸残基。从推测得到的氨基酸序列的比对分析得出：云南窗萤的荧光素酶与来自Lampyris noctiluca, L. turkestanicus和Nyctophila cf. caucasica三种萤火虫的荧光素酶有97.8%的序列一致性。从推测得出的氨基酸序列进行系统发育分析,其结果表明：云南窗萤和Lampyris+Nyctophila聚在一起, 与同属的发光强夜行性的萤火虫不形成的单系。云南窗萤荧光素酶在大肠杆菌中表达的条带大约70 kDa,并且在有荧光素存在时发出黄绿色荧光。对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点。云南窗萤和窗萤属的其他3种萤火虫的荧光素酶相比,有13个不同氨基酸位点,位于模拟分子结构的表面。对于这些多肽环、不同氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异。     

Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera)

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.     

Functional conversion of fatty acyl-CoA synthetase to firefly luciferase by site-directed mutagenesis: A key substitution responsible for luminescence activity

We demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis [Oba, Y., Ojika, M. and Inouye, S. (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett. 540, 251-254] and proposed that the evolutionary origin of beetle luciferase is a fatty acyl-CoA synthetase (FACS) in insect. In this study, we performed the functional conversion of FACS to luciferase by replacing a single amino acid to serine. This serine residue is conserved in luciferases and possibly interacts with luciferin. The mutants of FACSs in non-luminous click beetle Agrypnus binodulus (AbLL) and Drosophilamelanogaster (CG6178) gave luminescence enhancement, suggesting that the serine residue is a key substitution responsible for luminescence activity.     

Bioluminescence and chemiluminescence literature: 1987 literature,part III

In studying beetle bioluminescence in the early 1960s, Dr McElroy and his colleagues found that the Jamaican click beetle, Pyrophorus plagiophthalamus, was capable of emitting different colours of light. They further found that the luciferin substrate used by this beetle was the same as that in the firefly, demonstrating that the different colours of bioluminescence were due to differences in the structure of the luciferases. We have recently cloned cDNAs from this beetle species which code for at least four different luciferases. The luciferases are distinguishable by their different colours of bioluminescence when expressed in Escherichia coli. The sequence differences between these different luciferases are few, so the amino acids responsible for the different colours of emission must also be few. Through the construction of hybrid luciferases, by rearranging fragments of the original cDNA clones, we have identified some of these amino acid determinants of colour.     

Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods.

The results of the author's laboratory on the interaction of Luciola mingrelica firefly luciferase with substrates and their analogs using both steady-state and time resolved fluorescence are reviewed. The contribution of fluorescence of Trp and Tyr residues of the protein to its intrinsic fluorescence spectrum was estimated. Studies of quenching of Trp and Tyr fluorescence by luciferin and ATP allowed one to determine binding constants of the luciferase with substrates and to show that the binding of one substrate to the luciferase decreases the affinity of the enzyme for the other one. Fluorescence of oxyluciferin and its analogs (dimethyl- and monomethyloxyluciferins) was shown to be a good model of native firefly bioluminescence. A comparison of the fluorescence spectra of oxyluciferin and its analogs in aqueous solutions and in the presence of the luciferase revealed specific and nonspecific effects of the microenvironment on the equilibrium between different ionic forms of oxyluciferin. An approach based on photo-physical concepts of the correlation between luminescence spectra and structure of the emitter and its microenvironment was proposed and this approach was used to analyze bioluminescence spectra of wild-type and mutant luciferases.     

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey.

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin.