The orange light emitting luciferase from the rare Euryopa clarindae adult railroadworm (Coleoptera:Phengodidae): structural/functional and evolutionary relationship with green and red emitting luciferases.

Railroadworms luciferases emit the widest range of bioluminescence colors among beetles, ranging from green to red, being model enzymes to investigate the structure and bioluminescence colors relationships. Only three active railroadworms luciferases from the larval stage have been cloned and investigated: the Phrixothrix hirtus head lanterns red-emitting luciferase (PhRE); the Phrixothrix vivianii lateral lanterns green emitting luciferases (PvGR) and the Phengodes sp. dorsal lanterns yellow-green emitting luciferase (Ph). No active luciferase emitting in the yellow-orange region, however, has been cloned yet. Here we report the cloning and characterization of the orange emitting luciferase from the adult males of a rare Brazilian Cerrado railroadworm, Euryopa clarindae, and the transcriptional identification of two isozymes from the Amazon forest Mastinomorphus sp. railroadworm. The luciferase of E. clarindae has 548 residues, emits orange bioluminescence (600 nm), and displays intermediate kinetic values [KM(luciferin) = 50 µM, KM(ATP) ~ 170 µM] between those reported for green-emitting lateral lanterns and red emitting head lanterns luciferases. It displays 74-78% identity with the lateral lanterns luciferases of other railroadworms and 70% with the head lantern PhRE luciferase, and 96% with the larval Mastinomorphus sp. Mast-1, suggesting that this larva could be from the Euryopa genus. The phylogenetic analysis and kinetic/functional properties, place this orange-emitting enzyme as an intermediate form between the green-emitting lateral lanterns and red-emitting head lanterns luciferases. Major structural differences that could be associated with bioluminescence color determination are a relatively larger cavity size, and substitutions in the loops 223-235 and 311-316, especially N/C/T311, and their interactions which may help to close the bottom of LBS.

Cloning and molecular properties of a novel luciferase from the Brazilian Bicellonycha lividipennis (Lampyridae: Photurinae) firefly: comparison with other firefly luciferases.

Several firefly luciferases eliciting light emission in the yellow-green range of the spectrum and with distinct kinetic properties have been already cloned, sequenced, and characterized. Some of them are currently being applied as analytical reagents and reporter genes for bioimaging and biosensors, and more recently as potential color tuning indicators of intracellular pH and toxic metals. They were cloned from the subfamilies Lampyrinae (Photinini: Photinus pyralis, Macrolampis sp2; Cratomorphini: Cratomorphus distinctus), Photurinae (Photuris pennsylvanica), Luciolinae (Luciola cruciata, L. lateralis, L. mingrelica, L. italica, Hotaria parvula), and Amydetinae (Amydetes vivianii) occurring in different parts of the world. The largest number has been cloned from fireflies occurring in Brazilian biomes. Taking advantage of the large biodiversity of fireflies occurring in the Brazilian Atlantic rainforest, here we report the cloning and characterization of a novel luciferase cDNA from the Photurinae subfamily, Bicellonycha lividipennis, which is a very common firefly in marshlands in Brazil. As expected, multialignements and phylogenetic analysis show that this luciferase clusters with Photuris pennsylvanica adult isozyme, and with other adult lantern firefly luciferases, in reasonable agreement with traditional phylogenetic analysis. The luciferase elicits light emission in the yellow-green region, has kinetics properties similar to other adult lantern firefly luciferases, including pH- and metal sensitivities, but displays a lower sensitivity to nickel, which is suggested to be caused by the natural substitution of H310Y.

Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Role of E270 in pH- and metal-sensitivities of firefly luciferases.

Firefly luciferases display a typical change in bioluminescence color to red at acidic pH, high temperatures and in the presence of heavy metals. Recently, the proton and metal sensing site responsible for the pH-sensitivity of firefly luciferases, which involves the salt bridges between E311-R337 and H310-E354, was identified. However, it is unclear what other residues contribute to the distinct degrees of pH-sensitivity observed in other firefly luciferases. A multialignment of primary structures of a large set of pH-sensitive and pH-insensitive beetle luciferases showed that the conserved E270 among adult firefly luciferases is substituted by Gly (railroad worms)/Gln (click-beetles) in pH-insensitive ones. Site-directed mutagenesis studies using Macrolampis sp2 and Amydetes vivianii firefly luciferases indeed showed that E270 is important for the pH-dependent activity and spectral profiles: the substitution E270A/G drastically decreases the spectral pH-sensitivity, and extends the activity profile above pH 9.0. These mutations also decrease the sensitivity to metals such as zinc, mercury and cadmium. Modelling studies showed that the residue E270 is located in a three-glutamate motif (269EEE271) at the N-terminal of α-helix-10. The results suggest that at acidic pH, the protonation of E270 carboxylate may extend a turn of the helix at the N-terminal, misaligning the pH-sensor and luciferin phenolate binding site residues: S286, I288 and E311. In contrast, the substitution of E270A/G may unwind a turn of the α-helix-10, indirectly increasing the interaction of the pH-sensor and other residues at the bottom of the luciferin binding site, stabilizing the green light emitting conformation.

A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays.

Firefly luciferases have been widely used for bioanalytical purposes during the last 5 decades. They usually emit yellow-green bioluminescence and are pH-sensitive, displaying a color change to red at acidic pH and higher temperature and in the presence of heavy metals. Besides the usual applications as bioanalytical reagents and as reporter genes, firefly luciferases' pH- and metal-sensitivities have been recently harnessed for intracellular metal and pH biosensing. Previously we cloned the luciferase of the Brazilian Amydetes vivianii firefly which displays the most blue-shifted color among known firefly luciferases. Here we purified it, characterized and investigated the kinetic properties and the pH, metal and thermal sensitivities of this firefly luciferase. This luciferase displays the lowest reported KM for ATP, the highest catalytic efficiencies, and the highest thermostability among the studied recombinant beetle luciferases, making this enzyme and its cDNA an ideal reagent for sensitive ATP assays and reporter gene. The blue-shifted spectrum, higher thermostability, lower pH- and thermal-sensitivities and protein fluorescence studies indicate a more rigid active site during light emission. This enzyme displays an unmatched selective spectral sensitivity for cadmium and mercury, making it a promising ratiometric indicator of such toxic metals. Finally, the weaker thermal-sensitivity compared to other firefly luciferases makes this enzyme a better ratiometric pH indicator at temperatures above 30 °C, suitable for mammalian cell assays.

Phrixotrix luciferase and 6'-aminoluciferins reveal a larger luciferin phenolate binding site and provide novel far-red combinations for bioimaging purposes.

How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6'-substituted aminoluciferin analogues (6'-morpholino- and 6'-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the KM and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6'-morpholino- (634 nm) and 6'-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6'-aminoluciferins provide potential far-red combinations for bioimaging applications.

Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also providing a base to accept excited oxyluciferin phenol proton, and a countercation to shield the negative charge of E311 and to stabilize excited oxyluciferin phenolate, blue-shifting emission spectra in most beetle luciferases.

Preparation and Assay of Simple Light off Biosensor Based on Immobilized Bioluminescent Bacteria for General Toxicity Assays.

We describe a simple light off bioluminescent biosensor for general environmental toxicity assays based on immobilized bioluminescent bacteria engineered with beetle luciferases.

The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343).

First transcriptional survey of the Malpighian tubules of giant mealworm, Zophobas morio (Coleoptera: Tenebrionidae).

The Malpighian tubules play a key role in insect osmoregulation. Although a transcriptional analysis has been done for the Malpighian tubules in Drosophila melanogaster (Diptera), no functional genomics analysis has yet been carried out for any Coleoptera species. Recently, we constructed a cDNA library from Malpighian tubules of larval Zophobas morio, a close relative of Tribolium castaneum, and cloned the cDNA for an AMP/CoA-ligase with luciferase-like enzyme properties. Using this cDNA library, we randomly isolated, partially sequenced and analyzed ca. 540 clones, obtaining the first transcriptional profile of the most representative expressed genes, and associated them with their possible biological functions. A high percentage of mitochondrial genes was found, which is consistent with the high metabolic activity required by this organ during the formation of primary urine. Common transcripts included those for enzymes involved in osmoregulation, such as solute transporters and ATPases, and in detoxification and excretion, such as cytochrome P450, glutathione S-transferase, alcohol dehydrogenase. The presence of AMP/CoA-ligases, which activate exogenous carboxylic acids such as firefly D-luciferin suggests their participation in important new xenobiotic excretion/detoxification roles in Malpighian tubule physiology.

A transcriptional and proteomic survey of Arachnocampa luminosa (Diptera: Keroplatidae) lanterns gives insights into the origin of bioluminescence from the Malpighian tubules in Diptera.

Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera.

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor.

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae.

A route from darkness to light: emergence and evolution of luciferase activity in AMP-CoA-ligases inferred from a mealworm luciferase-like enzyme.

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

The luciferin binding site residues C/T311 (S314) influence the bioluminescence color of beetle luciferases through main-chain interaction with oxyluciferin phenolate.

Beetle luciferases emit different bioluminescence colors from green to red; however, no clear relationship between the identity of the luciferin binding site residues and bioluminescence colors was found in different luciferases, and it is unclear whether critical interactions affecting emission spectra occur on the thiazolyl or on the benzothiazolyl sides of the luciferin binding site. Through homology modeling and site-directed mutagenesis using our multicolor set of beetle luciferases (Pyrearinus termitilluminans larval click beetle, Pte, λ(max) = 534 nm; Phrixothrix hirtus railroad worm red emitting, PxRE, λ(max) = 623 nm; and Macrolampis sp2 firefly, Mac, λ(max) = 564 nm), we show that the residues C/T311 (S314) play an important role in bioluminescence color determination. Modeling studies indicate that the main-chain carbonyls of these residues are close to both oxyluciferin phenolate and AMP, whereas the side chains pack against second-shell residues. The C311(S314)A mutation considerably red shifts the spectra of the green-yellow-emitting luciferases (Pte λ(max) = 534 to 590 nm; Mac λ(max) = 564 to 583/613 nm) and affects the K(M) values for luciferin and ATP, but not the spectrum of the red-emitting luciferase. On the other hand, whereas the exchange between C/T311 (S314) caused smaller effects on the emission spectra of green-yellow-emitting luciferases, the C311T substitution (naturally found in green-emitting railroad worm luciferases) resulted in the largest reported blue shift in P. hirtus red-emitting luciferase (λ(max) = 623 to 606 nm). Altogether, these results indicate that the stability of residues C/T311 (S314) and the size of the cavity around oxyluciferin phenolate affect bioluminescence colors and suggest, for the first time, the occurrence of a critical interaction between main-chain carbonyls of position 311 (314) residues and oxyluciferin phenolate.

Aspisoma lineatum (Gyllenhal) (Coleoptera: Lampyridae) firefly: description of the immatures, biological, and ecological aspects.

Aspisoma lineatum (Gyllenhal) is a common firefly in the Southeastern region of Brazil. Adults and larvae were collected in the municipality of Campinas, state of São Paulo, Brazil, and the immature stages were described and reared in the laboratory. Four generations were reproduced in the laboratory, and a method for its rearing was established. The life cycle usually lasts 6 months, but under optimal laboratory conditions, it lasted from 2 to 4 months. Larvae were fed with Bradybaena similaris and Bulimulus tenuissimus snails since the beginning of the larval stage. This species was found to be easily adapted to environments under anthropic influence, such as urban areas and farms.

Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases.

Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, K(M) values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging.

An ancestral luciferase in the Malpighi tubules of a non-bioluminescent beetle!

The evolutionary origin of beetle bioluminescence is enigmatic. Previously, weak luciferase activity was found in the non-bioluminescent larvae of Tenebrio molitor (Coleoptera: Tenebrionidae), but the detailed tissular origin and identity of the luciferase-like enzyme remained unknown. Using a closely related giant mealworm, Zophobas morio, here we show that the luciferase-like enzyme is located in the Malpighi tubules. cDNA cloning of this luciferase like enzyme, showed that it is a short AMP-ligase with weak luciferase activity which diverged long ago from beetle luciferases. The results indicate that the potential for bioluminescence in AMP-ligases is very ancient and provide a first reasonable protoluciferase model to investigate the origin and evolution of beetle luciferases.

CCD imaging of basal bioluminescence in larval fireflies: clues on the anatomic origin and evolution of bioluminescence.

The anatomic and biochemical origin of beetle bioluminescence is still poorly understood. Through CCD imaging we report that larvae and pupae of the Brazilian fireflies Aspisoma lineatum and Cratomorphus sp emit a continuous weak glow throughout the entire body during all stages. This luminescence is especially developed after feeding, ecdysis and in the pupal stage, gradually disappearing as the cuticle becomes sclerotized and the adult emerges. This weak glow arises from the fat body, which consists of small lobes spread all over the body cavity. According to their pigmentation, these lobes can be divided in whitish and pinkish, and display different luciferase isozymes. Morphological studies suggest that the jelly-like ventral lanterns in the 8th abdominal segment evolved from these white lobes, providing a rationale for the widespread location of lanterns in larvae of different bioluminescent beetles. The biological and biochemical function of this weak diffuse bioluminescence is discussed in the context of the larval life-history.