Luciferin-regenerating Enzyme Research Articles

Realization of firefly bioluminescence cycle in vitro and in cells

The quantum yield (Q) of the “cold light” of firefly bioluminescence (BL) is remarkably high due to its nonradiative decay is extremely minimized. Thus, an artificial firefly represents the new generation of biomimetic “cold light” source with highest energy utilization. However, to manufacture a firefly-biomimetic “cold light” in vitro, one has to overcome several challenges including realization of the firefly BL cycle by incorporating the two important enzymes (i.e., firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE)) in one system. Here in this work, using self-prepared Fluc, LRE, and the main substrates, we realized the firefly BL cycle both in vitro and in cells. Moreover, using combinational analyses of HPLC and nESI-CID-MS/MS, we identified the main chemicals in the metabolic pathways underlying the firefly BL cycle. Using theoretical simulations, we revealed an optimum chemical route which balances the reaction cycle to achieve the highest BL intensity with the least chemical supplies. We anticipate that this pioneering study of the firefly cycle would provide industry with the opportunity to design tunable, economical, biomimetic “cold light” device in near future.

Bacteria-Based Live Vehicle for In Vivo Bioluminescence Imaging.

The anticancer therapy strategy mediated by tumor-targeting bacteria needs better visualization tools for imaging and monitoring bacteria in vivo. The probiotic strain Escherichia coli Nissle 1917 (EcN), one of the tumor-targeting bacteria, leads to the potential application for cancer therapy. Here, we report the development and application of a live, EcN-based imageable vehicle for noninvasive in vivo bioluminescence imaging in live mice. Firefly luciferase (Fluc) and luciferin-regenerating enzyme (LRE), an enzyme that contributes to stable bioluminescence, were functionally coexpressed in EcN. The recombinant EcN strain expressing the genomically integrated Fluc-LRE cassette was demonstrated to be a valuable tool for generating robust, continuous, and red-shifted bioluminescence for bacterial tracking in vitro and in vivo, thus providing an optical tumor-targeting system for the in vivo study of bacteria-assisted cancer therapy. Additionally, in vivo imaging of the recombinant EcN strain in the mouse intestinal tract indicated the potential of this strain to be used as a tool in the study of gut.

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis, Condensation and Chiral Inversion.

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.

Biosynthetic bifunctional enzyme complex with high-efficiency luciferin-recycling to enhance the bioluminescence imaging.

Firefly luciferase is a prominent reporter on molecular imaging with the advantage of longer wavelength on light emission and the ATP linear correlation, which makes it useful in most of current bioluminescence imaging model. However, the utility of this biomaterial was limited by the signal intensity and stability which are respectively affected by enzyme activity and substrate consumption. This study demonstrated a series of novel synthetic bifunctional enzyme complex of Firefly luciferase (Fluc) and Luciferin-regenerating enzyme (LRE). A peptide linker library was constructed for the fusion strategy on biosynthesis. The findings of both experimental data and structural simulation demonstrated that the intervention of fused LRE remarkably improve the stability of in vitro bioluminescence signal through luciferin recycling; and revealed the competitive relationship of Fluc and LRE on luciferin binding: Fluc performed higher activity with one copy number of rigid linker (EAAAK) at the C terminal while LRE acted more efficiently with two copy numbers of flexible linker (GGGGS) at the N terminal. With the advantage of signal intensity and stability, this fused bifunctional enzyme complex may expand the application of firefly luciferase to in vitro bioluminescence imaging.

Fusion expression of bifunctional enzyme complex for luciferin-recycling to enhance the luminescence imaging

Firefly luciferase (Fluc) has been widely used as a bioluminescent monitor. The ATP linear correlation and exogenous luciferin requirement make it useful in most of current imaging systems. However, the utility of this reporter was still limited by the intensity and decay of the luminescent signal, and the active site and structure of enzyme including the relevant substrate channeling region.This study demonstrated a novel construction of bifunctional enzyme system to improve the luminescence generation of firefly luciferase, by bringing in a luciferin-regenerating enzyme (LRE) fusion expressed to the C terminal of luciferase, between which were connected with peptide linker. The fusion protein constructed with typical type of linker, rigid linker (EAAAK) and flexible linker (GGGGS), were analyzed comparing with the unlinked free enzyme. In vivo and in vitro assessment of the bioluminescence intensity and decaying rate to the series of Fluc-LRE enzyme complex were assayed. The fInding demonstrated that the presence of LRE remarkably enhance the generation of luminescence and remained significant stronger signal than that of the control, and the peptide-linked dual enzyme present more stability and continuation on the signal generation and lower decaying rate on signal recession, especially at low dose of Fluc injection. With the advantage of luminescence intensity and reaction period, the peptide mediated fusion expressed LRE may expand the application of Firefly luciferase on bioluminescence imaging.

Transcriptomes from the photogenic and non-photogenetic tissues and life stages of the Aspisoma lineatum firefly (Coleoptera: Lampyridae): Implications for the evolutionary origins of bioluminescence and its associated light organs

In fireflies, the bioluminescent system involves a benzothiazolic luciferin, ATP, a luciferase, and complex lanterns consisting of different tissues including a photogenic one. Whereas the biochemical reaction and the structure of lanterns are quite well known, the evolutionary and developmental origins of photogenic tissue as well as the biosynthetic origin of luciferin remain enigmatic. One approach to better understand the origin of the photogenic tissue and biosynthetic origin of luciferin is to investigate the molecular differentiation among photogenic and non-photogenic tissues. Thus, here we compared - for the first time - the transcriptomes of the photogenic tissue of lanterns (specialized tissue) and the fat body (non-specialized tissue) of larvae, adult lanterns, and eggs stages of the common Southeastern Brazilian firefly Aspisoma lineatum. The similarities between the photogenic tissue of lanterns and the fat body, with the occurrence of several luciferase isozymes, luciferin-regenerating enzyme, L-DOPA metabolism related enzymes, and nitric oxide synthase, support the hypothesis that in fireflies, the photogenic tissue evolved from the fat body.

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.

Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography.

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

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 (T(69)R) was selected for this research and the effects of D- and L-cysteine on T(69)R T-LRE-luciferase-coupled assay are examined. Our results demonstrate that bioluminescent signal of T(69)R 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 T(69)R 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.

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 T(69)R 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 (K(77)E/T(69)R) increased the amount of bioluminescent signal more than two-fold over a short time. In addition, G(75)E, K(77)E and G(75)E/T(69) R mutants did not improve luciferin-luciferase in vitro bioluminescence. Based on our results, addition of K(77)E/G(75)E and K(77)E/G(75)E/T(69)R 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.

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.

Structure and evolution of the luciferin-regenerating enzyme (LRE) gene from the firefly Photinus pyralis.

To study the structural features of genes for the luciferin-regenerating enzyme (LRE), the entire gene along with 524 bp of upstream sequence was determined from Photinus pyralis (Coleoptera: Lampyridae). The LRE gene revealed an open reading frame composed of five exons divided by four introns ranging in size from 47 to 904 bp. The deduced LRE amino acid sequence showed identity to senescence marker protein-30 (SMP30) from a number of insects and mammals including four putative SMP30 sequences from Anopheles gambiae. Gene structure comparisons showed some intron/exon site conservation with A. gambiae and mammalian SMP30 proteins but not Drosophila. LRE and luciferase sequence comparisons revealed two conserved putative luciferin-binding sites. The evolution of LRE was discussed in relation to its function.

Molecular cloning and expression of the cDNAs encoding luciferin-regenerating enzyme from Luciolacruciata and Luciolalateralis

In the firefly light organ, oxyluciferin, a product of the light-emitting reaction of firefly luciferase, is thought to be converted into luciferin. Previously, we isolated the luciferin-regenerating enzyme (LRE) from Photinuspyralis. LRE plays an important role in the recycling of oxyluciferin into luciferin. We have cloned two cDNAs encoding LRE, G-LRE and H-LRE, from poly(A)+ RNA of the lanterns of Luciolacruciata and Luciolalateralis, using reverse transcription–polymerase chain reaction, 5′-RACE (5′-rapid amplification of cDNA ends) and 3′-RACE. The putative translation products have molecular masses of 33,804 and 34,285 Da, corresponding to 309 and 307 amino acids, respectively. The deduced amino acid sequence of G-LRE shows 57 and 56% identity with H-LRE and A-LRE (P. pyralis), respectively. LRE (G-LRE, H-LRE, A-LRE) shows at most 39% amino acid sequence identity with insect anterior fat protein (AFP) and mammalian senescence marker protein-30 (SMP30). G-LRE and H-LRE were successfully expressed under the control of the lac promoter in Escherichiacoli.

Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin

The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.