Lampyris Turkestanicus Research Articles

Genomic and protein structure analysis of the luciferase from the Iranian bioluminescent beetle, Luciola sp.

To date, two Iranian luciferase genes from the Lampyris turkestanicus and Lampyroidea maculata have been carefully studied. Here, we report the cloning and characterization of the gene and protein of luciferase enzyme from the beetle of an Iranian lampyrid species, Luciola sp. (Coleoptera-Lampyridae). In this study, a Luciola sp. firefly was collected from the Yasouj area of Iran and its luciferase gene sequence was cloned and characterized. The genomic DNA length for this luciferase was the 1950 bp that combined of seven exons and separated by six introns. The results of multiple sequence alignment show that this gene has the most similarity with DNA gene luciferase from the Hotaria unmunsana species. Further analysis determined accurately the location of these introns in the luciferase gene. However, the deduced amino acid sequences of the luciferase gene (548 residues) showed that this luciferase had 97.8% resemblance to luciferase from Lampyroidea maculata species. By in silico modeling of firefly luciferase in an I-TASSER server, the 3D structure of this enzyme was evaluated. The results of phylogenetic tree analysis display the close evolutionary relationship of this luciferase gene and luciferase gene from the Lampyroidea maculata and Hotaria unmunsana.

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

Contribution of luciferin-regenerating enzyme (LRE) for invitro recycling of D-luciferin has been reported. According to crystal structure of LRE, it is abeta-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.

Surface charge modification increases firefly luciferase rigidity without alteration in bioluminescence spectra.

Protein engineering can provide useful approaches for loop anchoring and mutation of surface-exposed loop residues to Arg for the design of thermostable proteins. In this context and due to the high proportion of surface loops, some of the solvent-exposed residues in the Lampyris turkestanicus luciferase were mutated to Arg. Using the red-emitter mutant luciferase (E354R/Arg356), the single (-Q35R, -I182R, -I232R and -L300R), double (-Q35R/I232R) and triple (-Q35R/I232R/I182R) mutant luciferases were introduced. The relative remaining activity of -I232R, double and triple mutants increased significantly compared to the wild-type at 40°C. The optimal temperature of these mutants increased up to 40°C which were 15°C more than wild-type luciferase. It is anticipated that these mutations increased the local interactions that finally improved the thermostability and optimum temperature of luciferase. It should be noted that Arg substitution at amino acid positions 35, 182 and 232 had no effect on the bioluminescence emission spectra. Furthermore, these mutations have not significantly changed the specific activities of firefly luciferases. Finally, with the use of the homology modeling and molecular docking, the effects of these substitutions were evaluated. In conclusion, this study provides beneficial insights on how the thermal stability of luciferase can be improved by protein engineering for biological applications.

Surface Arginine Saturation Effect on Unfolding Reaction of Firefly Luciferase: A Thermodynamic and Kinetic Perspective.

Replacement of some hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase with arginine increases thermostability of this enzyme. Herein, thermodynamic and kinetic of unfolding reactions of wild type (WT), E354R/356R, E354R/356R-I232R and E354R/356R-Q35R/L182R/I232R variants, has been investigated. Fluorescence and Far-UV circular dichroism measurements using urea as a chemical denaturant indicated that the value of ΔG(H2O) for all variants is greater than that of WT enzyme. Analysis of m-values, as a measure of difference in the solvent accessible surface area between the native and denatured states of protein, revealed that higher stability of mutants is related to their higher degree of compactness in the folded state. Results of unfolding kinetic experiments showed that all variants have three-exponential behavior in which they unfolded with three rate constants and corresponding amplitudes. Increasing the rate constants of fast unfolding phase in mutants relative to WT protein may be attributed to more compactness and more kinetic sensitivity of their folded state to urea. However, more population of WT protein was unfolded from fast unfolding phase. Results of this investigation highlight kinetic stability of luciferase via a slow rate of unfolding.

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift

Firefly bioluminescence reaction in the presence of Mg2+, ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350–359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351–359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.

Design of thermostable luciferases through arginine saturation in solvent-exposed loops

In most bioluminescence systems the oxidation of luciferin and production of light is catalyzed by luciferases. Protein engineering studies have shown that thermostable proteins from thermophilic organisms have a higher frequency of Arg, especially in exposed states. To further clarify the arginine saturation effect on thermostability of firefly luciferase, some of hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase are changed to arginine. All of these residues are located at the surface loops of L.turkestanicus luciferase. Starting with a luciferase mutant (E³⁵⁴Q/Arg³⁵⁶), single mutation (-Q³⁵R, -I¹⁸²R, -I²³²R and -L(300)R), double mutation (-Q³⁵R/I²³²R) and triple mutation (-Q³⁵R/I²³²R/I¹⁸²R) are added. Bioluminescence emission spectra indicate that substitution of Arg by these residues, do not effect on the maximum wavelength of emission spectrum. It should be noted, introduction of double mutation (-Q³⁵R/I²³²R) and triple mutation (-Q³⁵R/I²³²R/I¹⁸²R) were kept specific activity of firefly luciferase. By addition of positively charged residue, some specific mutations (-I²³²R, -Q³⁵R/I²³²R and -Q³⁵R/I²³²R/I¹⁸²R) showed that optimum temperature of activity was increased to 40°C which are 12 and 15°C higher than E³⁵⁴Q/Arg³⁵⁶ and wild-type luciferases, respectively. Also, after 40 min incubation of enzymes at 40°C, the relative remaining activity of wild type was only 5%, whereas for -I²³²R, -Q³⁵R/I²³²R and -Q³⁵R/I²³²R/I¹⁸²R was 60, 80 and 80% of original activity, respectively.

Role of charge distribution in a flexible loop on bioluminescence color of firefly luciferase: a lesson from nature

The presence of Arg353 in PhRE luciferase, which corresponds to the deleted residue in green emitter luciferases, is an important distinctive structural feature of it. Insertion and substitution of positively‐charged amino acids in a flexible loop (352TPEGDDKP359) of firefly luciferase brought about with change of bioluminescence color. Insertion of Arg356 in a green‐emitter luciferase (Lampyris turkestanicus), that is corresponding to Arg353 in P. hirtus, changed the emitted light from green to red. In order to further clarification of the effect of this position in the light shift, residues with positive side‐chain (Arg356 and Lys356) were inserted in Photinus pyralis luciferase which changed the light color to red, while insertion of a residue with negative side‐chain (Glu356) had little effect and a neutral residue (Gln356) had not any effect on color. On the other hand, by insertion and substitution of positive‐charged residues, in the same loop of Lampyris turkestanicus luciferase, different mutation (E354R/Arg356, E354k/Arg356, E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of Arg356 produces a luciferase that emit red light with a single peak bioluminescence spectrum.

RACE-based amplification of cDNA and expression of a luciferin-regenerating enzyme (LRE): An attempt towards persistent bioluminescent signal

In the firefly light organ, luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin. In this study, the LRE cDNA was cloned from total RNA extracted from lantern of Lampyris turkestanicus (an Iranian firefly) using PCR-based techniques including reverse transcription-polymerase chain reaction, 5′-RACE (5′-rapid amplification of cDNA ends) and 3′-RACE. The full-length cDNA encoding LRE of L. turkestanicus (T-LRE) was 1047 with 924 bp open-reading frame encoding the protein of 307 amino acid residues with a calculated molecular mass of 33.5 kDa. The deduced amino acid sequence of T-LRE showed 46 and 45% identity to A-LRE (LRE from the American firefly – Photinus pyralis) and G-LRE (LRE from a Japanese firefly – Luciola cruciata), respectively. The cDNA encoding LRE of L. turkestanicus was expressed in recombinant Escherichia coli and it is shown that T-LRE improves the luminescent signal of firefly luciferase.

Relationship between stability and bioluminescence color of firefly luciferase

Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.

Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study

Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg2+. Luciferase has a broad range of applications for analytical purposes and in vivo imaging for nanosystem biology studies. However, the enzyme is highly susceptible to proteolytic degradation that reduces its half-life. Rate of proteolytic digestion between two members of luciferase family (Photinus pyralis and Lampyris turkestanicus) is compared. Proteolytic sensitivity of L. turkestanicus luciferase was found to be more than P. pyralis luciferase, due to higher rate of hydrolysis under identical conditions. Both luciferases showed more sensitivity to chymotrypsin than trypsin with different digestion pattern. Digestion of P. pyralis by trypsin produced some fragments which were found to be more resistant to further degradation, whereas in L. turkestanicus initial fragments subdigested by trypsin, like chymotrypsin effect on both luciferases. Furthermore, both luciferases become increasingly labile to proteolysis as the temperature increases. The rate of inactivation and the rate of degradation between luciferases were different in a specific time of incubation. Appearance of similar bands for both luciferases confirmed exposure of specific regions, in spite of structural differences.

The effective role of positive charge saturation in bioluminescence color and thermostability of firefly luciferase

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Amongst different beetle luciferases, those from phrixothrix rail-road worm with a unique additional residue (Arg353) emit red bioluminescence color naturally. Insertion of Arg(356) in Lampyris turkestanicus luciferase changed the emitted light to red with a bimodal bioluminescence spectrum. By insertion and substitution of positively-charged residues, different specific mutation (E354R/Arg(356), E354K/Arg(356), E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of Arg(356) produces a luciferase that emits red light with a single peak bioluminescence spectrum. The comparison of mutants with native luciferase shows that mutations of firefly luciferase resulted in structural and functional thermostability. Comparative study of native and mutant luciferase (E354R/Arg(356)) by intrinsic and extrinsic fluorescence, CD spectropolarimetry, and homology modeling revealed mutation brought about an increase in content of secondary structure and globular compactness of L. turkestanicus luciferase. On the other hand, pK(a) of amino acids in the flexible loop decreased upon introducing of positive charges.

Effect of Charge Distribution in a Flexible Loop on the Bioluminescence Color of Firefly Luciferases

Firefly luciferase is a monooxygenase that catalyzes the ATP-dependent conversion of firefly luciferin into a luciferyl-adenylate, which is oxidized to an electronically excited oxyluciferin in a multistep reaction and produces visible light with a remarkable quantum yield. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetles, only luciferase from Phrixotrix railroad worm (Ph(RE)) emits red bioluminescence, naturally. The presence of Arg353 in Ph(RE) luciferase, which corresponds to the deleted residue in the other luciferases, is an important distinctive structural feature of it. Insertion of Arg356 into a green-emitter luciferase (Lampyris turkestanicus), corresponding to Arg353 in Phrixotrix hirtus, changed the emitted light from green to red. To further clarify the effect of this position on the light shift mechanism, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase, using site-specific insertion mutagenesis. Insertion of a residue with a positive side chain (Arg356 and Lys356) changed the light color to red, while insertion of a residue with a negative side chain (Glu356) had little effect on color. Insertion of a neutral residue (Gln356) at this position was performed without any change in bioluminescence spectra. Insertion of positively charged residues in this loop took place with a series of structural changes which were confirmed by fluorescence spectroscopy and homology modeling. Homology modeling reveals the appearance of a bulge in a flexible loop (T352-P359) upon mutation which shifts to the left side with a color change from green to red.

Cloning,Expression and Sequence Analysis of A Luciferase Gene from the Chinese Firefly Pyrocoelia pygidialis

The cDNA encoding the luciferase from lantern mRNA of one diurnal firefly Pyrocoelia pygidialis Pic, 1926 has been cloned, sequenced and functionally expressed. The cDNA sequence of P. pygidialis luciferase is 1647 base pairs in length, coding a protein of 548 amino acid residues. Sequence analysis of the deduced amino acid sequence showed that this luciferase had 97.8% resemblance to luciferases from the fireflies Lampyris noctiluca , Lampyris turkestanicus and Nyctophila cf. caucasica . Phylogenetic analysis using deduced amino acid sequence showed that P. pygidialis located at the base of Lampyris+Nyctophila clade with robust support (BP=97%); but did not show a monophyletic relationship with its congeneric species P. pectoralis, P. rufa and P. miyako, all three are strong luminous and nocturnal species. The expression worked in recombinant Escherichia coli. Expression product had a 70 kDa band and emitted yellow-green luminescence in the presence of luciferin. Five loops in the P. pygidialis luciferase, L1 (N198-G208), L2 (T240-G247), L3 (G317-K322), L4 (L343-I350) and L5 (G522-D532), were found from the structure modeling analysis in the cleft, where it was considered the active site for the substrate compound entering and binding. Different amino acid residues between the luciferases of P. pygidialis and the three other known strong luminous species can not explain the situation of weak or strong luminescence. Future study of these loops, residues or crystal structure analysis may be helpful in understanding the real differences between the luciferases between diurnal and nocturnal species.

Spectroscopic and functional characterization of <italic>Lampyris turkestanicus</italic> luciferase: a comparative study

Functional expression and spectroscopic analysis of luciferases from Lampyris turkestanicus and Photinus pyralis were carried out. cDNA encoding L. turkestanicus luciferase was isolated by reverse transcription-polymerase chain reaction, cloned, and functionally expressed in Escherichia coli. The luciferases were purified to homogeneity using Ni-nitrilotriacetic acid Sepharose, and kinetic properties of luciferase from L. turkestanicus were compared with that from P. pyralis. Amino acid differences in its primary structures in relation to P. pyralis luciferase brought about changes in the kinetic properties of the enzyme as evidenced by substantial lowering of Km for ATP, increased light decay time, and decreased thermostability. Luciferase from L. turkestanicus was used to carry out Michaelis-Menten kinetics with a Km of 95.5 muM for ATP and 20 muM for luciferin. Maximum activity was recorded at pH 8.5, so it might be a suitable reporter for microbial screening at alkaline pH. Tryptophan fluorescence for P. pyralis luciferase was higher than L. turkestanicus luciferase. Substitution of some residues in L. turkestanicus luciferase appears to change the kinetic properties by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by intrinsic and extrinsic fluorescence, Fourier transform infrared spectroscopy, and near-ultraviolet circular dichroism spectra.

Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases

The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K(m) and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

The Influence of Insertion of a Critical Residue (Arg356) in Structure and Bioluminescence Spectra of Firefly Luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Molecular cloning, sequence analysis, and expression of a cDNA encoding the luciferase from the glow-worm, Lampyris turkestanicus

The first cDNA from lampyridae encoding a glow-worm luciferase from lantern mRNA of Lampyris turkestanicus has been cloned, sequenced, the amino acid sequence predicted, and the sequence reported to GenBank. The cDNA was 1644 base pairs in length and coding a 547-residue protein. The deduced amino acid sequence of the luciferase gene of L. turkestanicus showed 98.7% and 95.8% identity to Lampyris noctiluca and Pyrocoelia rufa, respectively. Phylogenetic analysis further confirmed that the deduced amino acid sequences of L. turkestanicus luciferase gene belong to the same subfamily, Lampyrinae. The cDNA encoding the luciferase of L. turkestanicus was expressed as a 62kDa band in recombinant Escherichia coli and showed green luminescence in the presence of luciferin. Amongst amino acid differences of L. turkestanicus and L. noctiluca (its clade) there are two important substitutions. Signature amino-acid sequences and motifs found in the deduced sequence are CK2-phospho site, ASN-glycosylation, myristoylation site, PKC-phospho site, microbodies C-terminal targeting signal, and AMP-binding domain.

Separation, identification and determination of luciferin in the Iranian firefly, Lampyris turkestanicus by HPLC and spectroscopic methods.

Iranian firefly larvae, Lampyris turkestanicus collected from north of Iran and their luciferin were analyzed by HPLC, TLC, MS and spectroscopic Fourier transform ([FT]-IR, FT-NMR, UV and fluorescence) methods. The results showed that luciferin in L turkestanicus lanterns was the same as in the American firefly, Photinus pyralis and synthetic D-luciferin.