Comparison of Acyl-CoA Synthetic Activities and Enantioselectivity toward 2-Arylpropanoic Acids in Firefly Luciferases

Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the k _cat parameter contributed to the preferential enantioselectivity of this enzyme.     

Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues

We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.     

Purification and characterization of luciferases from fireflies, Luciola cruciata and Luciola lateralis.

Luciferases of Luciola cruciata and Luciola lateralis, LcL and LlL, were purified to homogeneity by ammonium sulfate precipitation, gel-filtration column chromatography, and hydroxyapatite HPLC. The molecular masses of the enzymes determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were both 62 kDa, almost identical to that of Photinus pyralis (PpL). LcL was found to be similar to PpL in thermal stability, pH stability, and the wavelength of maximum light intensity. LlL was superior to LcL and PpL in thermal and pH stability, and the reaction catalyzed by LlL emits green light with a peak intensity at 552 nm, which is 10 nm shorter in wavelength than those of PpL and LcL.     

New application of firefly luciferase--it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid

We introduce a new application of firefly luciferase (EC 1.13.12.7). The firefly luciferases belong to a large superfamily that includes rat liver long-chain acyl-CoA synthetase (LACS1). LACS1 is the enzyme that is involved in the deracemization process of 2-arylpropanoic acid and catalyzes the enantioselective thioester formation of R-acids. Based on the similarity of the reaction mechanisms and the sequences between firefly luciferase and LACS1, we predicted that firefly luciferase also has thioesterification activity toward 2-arylpropanoic acid. From an investigation using three kinds of luciferases from North American firefly and Japanese fireflies, we have confirmed that these luciferases exhibit an enantioselective thioester formation activity and the R-form is transformed to a thioester in preference to the S-form in the presence of ATP, Mg(2+), and CoASH. The enantiomeric excesses of unreacted recovered acid and thioester were determined by chiral phase HPLC analysis and the resulting 2-arylpropanoyl-CoAs were identified by high resolution mass spectroscopy. The K(m) and k(cat) values of thermostable luciferase from Luciola lateralis (LUC-H) toward ketoprofen were determined as 0.22 mM and 0.11 s(-1), respectively. The affinity of ketoprofen was almost the same of d-luciferin. In addition, the calculated E-value toward ketoprofen was approximately 20. These results suggest that LUC-H could catalyze the kinetic resolution of 2-arylpropanoic acid efficiently and would be a new option for the preparation of optically active 2-substituted carboxylic acids.     

Enantiodifferentiation of ketoprofen by Japanese firefly luciferase from Luciola lateralis

Recently, we found that firefly luciferase exhibited (R)-enantioselective thioesterification activity toward 2-arylpropanoic acids. In the case of Japanese firefly luciferase from Luciola lateralis (LUC-H), the E-value for ketoprofen was approximately 20. In this study, we used a spectrophotometric method to measure the catalytic activity of LUC-H. Using this method allowed us to judge the reaction efficiency easily. Our results confirmed that LUC-H exhibits enantioselective thioesterification activity toward a series of 2-arylpropanoic acids. The highest activity was observed with ketoprofen. We also observed high enzymatic activity of LUC-H toward long-chain fatty acids. These results were reasonable because LUC-H is homologous with long-chain acyl-CoA synthetase. To obtain further information about the enantiodifferentiation mechanism of the LUC-H catalyzed thioesterification of ketoprofen, we determined the kinetic parameters of the reaction relative to each of its three substrates: ketoprofen, ATP, and coenzyme A (CoASH). We found that whereas the affinities of each compound are not affected by the chirality of ketoprofen, enantiodifferentiation is achieved by a chirality-dependent difference in the k_cat parameter.     

Thr226 is a key residue for bioluminescence spectra determination in beetle luciferases

The comparison of click beetle and railroadworm luciferases (pH-insensitive) with firefly luciferases (pH-sensitive) showed a set of conserved residues differing between the two groups which could be involved with the bioluminescence spectra pH sensitivity. The substitution C258V in Pyrocoelia miyako (Pml) firefly luciferase and V255C in Ragophthalmus ohbai railroad worm luciferase (Rol) had no effect on the bioluminescence spectra. Substitution of Thr226 in the green-light-emitting luciferases of Rol and Pyrearinus termitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red-light-emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (lambda(max) = 611 nm). The bioluminescence spectrum of all these luciferase mutants did not show altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wild-type luciferases. Altogether present data suggest that Thr226 is an important residue for keeping active-site core in both groups of beetle luciferases. The mechanism for bioluminescence color determination between pH-sensitive and pH-insensitive luciferases could be different.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Mutant Luciferase Enzymes from Fireflies with Increased Resistance to Benzalkonium Chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.     

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.     

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.     

Bioluminescence Spectra of Native and Mutant Firefly Luciferases as a Function of pH

Bioluminescence spectra of the wild-type recombinant Luciola mingrelica firefly luciferase and its mutant form with the His433Tyr point mutation were obtained within the pH 5.6-10.2 interval. The spectra are shown to be a superposition of the spectra of the three forms of the electronically excited reaction product oxyluciferin: ketone (lambdamax = 618 nm), enol (lambdamax = 587 nm), and enolate-ion (lambdamax = 556 nm). The shift in lambdamax by 40 nm to the red region in the mutant luciferase bioluminescence at the pH optimum of enzyme activity (pH 7.8) is explained by the change in the relative content of different oxyluciferin forms due to the shift in the ketone <--> enol <--> enolate equilibria. A computer model of the luciferase-oxyluciferin-AMP complex was constructed and the structure of amino acid residues participating in the equilibrium is proposed. Computer models of the protein region near the His433 residue for the wild type and mutant luciferases are also proposed. Comparison of the models shows that the His433Tyr mutation increases flexibility of the polypeptide loop that binds the N and C domains of luciferase. As a result, the flexibility of the C domain amino acid residues in the emitter microenvironment increases, and this increase may be the reason for the observed differences in the bioluminescence spectra of the native and mutant luciferases.     

Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity

Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of bioluminescence spectra, and Y35N,H and F16L/A40S substitutions resulted in the enzymes with bioluminescence spectra virtually independent from pH in the range of 6.0-7.8. The structural explanation is proposed for the effect of mutations on pH-sensitivity of bioluminescence spectra.     

Codon-optimized Luciola italica luciferase variants for mammalian gene expression in culture and in vivo

Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-d-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.     

Luciferase cDNA from Japanese firefly,Luciola cruciata: Cloning,structure and expression in Escherichia coli

We have cloned the cDNA for luciferase from lantern poly(A)⁺ RNA of a Japanese firefly, Luciola cruciata (Genji botaru in Japanese). This cDNA directed the synthesis of enzymatically active luciferase under the control of the lac promoter in Escherichia coli. The amino acid sequence predicted from the cDNA sequence shows that Genji firefly luciferase consists of 548 amino acids and has a molecular weight of 60,024. Considerable sequence homology was found upon the comparison of the Genji and North American firefly luciferases.     

Trigger factor assists the refolding of heterodimeric but not monomeric luciferases

The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.     

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.     

Isolation and characterization of luciferase from Indian firefly,Luciola praeusta

Luciferase from Indian firefly Luciola praeusta (Coleoptera: Lampyridae: Luciolinae) was isolated and the properties compared with that of the North American firefly, Photinus pyralis. Luciola praeusta luciferase was purified using acetone extraction, gel‐filtration column chromatography, ammonium sulfate precipitation and anion exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis indicates a homogeneous preparation and the molecular mass was slightly higher than that of Photinus pyralis. The effect of pH, buffer composition and metal ions on the spectral characteristics was studied. The maximum bioluminescence activity of luciferase was observed in ACES buffer at pH 6.5. The emission maximum of 562 nm (in crude extract) was red shifted to 570 nm in Tricine buffer at pH 7.8. In addition, the effect of bovine serum albumin on the storage stability of the protein was investigated. Based on the unique spectral characteristics observed, we propose that Luciola praeusta luciferase in the native form is suitable for the assay of biochemical metabolites in acidic pH. Copyright © 2013 John Wiley & Sons, Ltd.     

Isolation and characterization of mutants of firefly luciferase which produce different colors of light

The luciferase cDNA from the 'Genji' firefly, Luciola cruciata, was mutated with hydroxylamine to isolate mutant luciferases. Some of the isolated mutant enzymes produced different colors of light, ranging from green to red. Five such mutants, producing green (lambda max = 558 nm), yellow-orange (lambda max = 595 nm), orange (lambda max = 607 nm) and red light (lambda max = 609 and 612 nm), were analyzed. The mutations were found to be single amino acid changes, from Val239 to Ile, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser and His433 to Tyr respectively.