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Keywords:

  • bioluminescence;
  • luciferase;
  • acyl-CoA synthetase;
  • L-luciferin;
  • D-luciferin;
  • oxyluciferin;
  • dehydroluciferyl-adenylate

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH2), with ATP-Mg2+ generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH2-AMP); the second is the oxidation and decarboxylation of LH2-AMP to oxyluciferin, the light emitter, producing CO2, AMP, and photons of yellow-green light (550– 570 nm). In a dark reaction LH2-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH2-CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3′-phosphate chain plus an intact terminal pyrophosphate moiety. The LH2 stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH2 or L-LH2, has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging. © 2008 IUBMB IUBMB Life, 61(1): 6–17, 2009


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

Bioluminescence, the result of a process that occurs in living organisms in which an electronically excited substance produced in a chemical reaction decays to the ground level1, is a widespread phenomenon in Nature. The scientific research on this subject led to the discovery of the enzyme-substrate system, the former called luciferase and the latter luciferin (from the Latin Lucifer, “Light-bringer”). Among all the bioluminescent organisms fireflies are the most studied and well-characterized, specially the North American firefly, Photinus pyralis (Order Coleoptera, Family Lampyridae).

The purpose of this review is to present the chemical reactions catalyzed by Photinus pyralis luciferase, furnishing basic information about its principal components, luciferase and luciferin. Also, some recent applications of the enzyme will be summarized together with well-established methods.

FIREFLY LUCIFERASE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

Firefly luciferase is classified as Photinus-luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing) (EC 1.13. 12.7). Its substrate is firefly luciferin (LH2), and the reaction requires ATP, oxygen and a metallic cation2, 3. Crystallographic studies have shown that the protein is folded into two compact domains, a large N-terminal domain and a small C-terminal domain, joined by a flexible linker peptide, which creates a wide cleft between the two domains [Fig. 1;4]. The C-terminal portion bears the tripeptide Serine-Lysine-Leucine (SLK motif) responsible for peroxisome targeting4, 5. The putative active site is believed to enclose amino acid residues on the surface of both domains, which suggests that during the course of the reaction the two domains will come together and cluster the substrate between them, requiring a significant conformational change4. Indeed, studies with wild-type and red mutant luciferase from Luciola cruciata showed that native luciferase adopts a “closed form” during the formation of the high-energy intermediate responsible for light emission, creating a hydrophobic pocket around the active site, and an “open form” in complex with the reactants and products6. The major part of the amino acid residues important to the bioluminescent activity is located in the N-terminal portion and only one in the C-terminal domain7, and it was demonstrated that luciferase can produce light bearing only the N-domain, albeit with a luminescent output of only 0.03% of the complete enzyme8.

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Figure 1. Photinus pyralis luciferase (Protein Data Bank accession number 1LCI) without its substrate in spacefill display. The image depicts the large N-terminal domain (N) and the small C-terminal domain (C), linked by a short loop (L). In this side-view representation the cleft between the two domains is visible. The image was generated using the program Rasmol version 2.7.3 (Raswin Molecular Graphics©).

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The typical emission spectrum for luciferase is in the yellow-green region (550–570 nm), with a peak at 562 nm at basic media (pH ∼ 7.5–7.8)9. However, luciferase is a pH-sensitive enzyme, and acid media (pH ∼ 5–6) can shift the emission to red (maximum at 620 nm), as well as higher temperatures and heavy metal cations10. It is believed that conformational changes, which influence the active site microenvironment, are responsible for the different color emission6. In Luciola cruciata red mutant luciferase the catalytic state remains in an “open form,” which could allow energy loss from the excited state intermediate, leading to the emission of red light instead of the higher-energy yellow-green light6.

The in vitro emission of light follows, under well-defined conditions, a flash pattern, with a rise in the intensity of emission that decays to low levels (about 5% of the initial burst) in a few seconds, even in the presence of available substrate [Fig. 2;3]. This profile was attributed to the formation of inhibitory products in the course of the reaction11, 12, a topic to be discussed later.

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Figure 2. In vitro profile of light production by luciferase with high substrate concentrations (D-LH2 and ATP-Mg2+ in the μM range). After a rapid increase in the light output to a maximum an accentuated decay in the intensity of emission is verified. (RLU, relative light unit).

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Firefly luciferin (LH2) has the chemical formula [(S)-2-(6′-hydroxy-2′-benzothiazolyl)-2-thiazoline-4-carboxylic acid] [Fig. 3; (13–15)], which was proposed and confirmed on the basis of chemical synthesis13 and X-ray crystallography16. In Nature, however, the biochemical pathway for LH2 synthesis remains a mystery15. In fact it is not even known if the fireflies contain all enzymes to synthesize LH2 or if it is obtained by ingestion of other bioluminescent organisms or by symbiosis with bacteria15. Also, the evolutionary creation of LH2 is questioned. Some researchers propose that LH2 first served as an antioxidant, later being requested by the bioluminescent reaction when luciferase appeared17. It has been suggested that the origin of the thiazoline ring could be a cysteine15, as can be observed in the chemical synthesis in which one of the steps involves this amino acid18. Cysteine has two isomers, L- and D-, and either one of them can be used to form LH2, giving rise to L-LH2 and D-LH2, respectively. Their function in the bioluminescent reaction will be highlighted in the incoming sections.

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Figure 3. Chemical structure of firefly luciferin, D-LH2. The two principal moieties of the molecule are shown, with the atoms numbering. Figure adapted from ref.15.

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FIREFLY LUCIFERASE REACTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

The bioluminescent reaction starts with the reaction of D-LH2 with ATP, as expressed in Eq. (1) [Fig. 4;19–22].

  • equation image(1)
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Figure 4. Chemical mechanism of the adenylylation of D-LH2. The reaction involves the displacement of inorganic pyrophosphate (PPi) as a leaving group from the ATP molecule, which leads to the formation of the intermediate D-luciferyl-adenylate (D-LH2-AMP).

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The reaction is a SN2 nucleophilic displacement, which involves the carboxylic group at the C4 carbon at the thiazoline ring of D-LH2 and the phosphate groups of ATP. The oxygen in the carboxyl group is a nucleophile, and nucleophilic attack of the electrophilic phosphorus at the α-phosphoryl group of ATP displaces inorganic pyrophosphate (PPi), an excellent leaving group, and transfers adenylate (5′-AMP) to D-LH2. The reaction is, thus, an adenylylation, and produces an enzyme-bound intermediate, luciferyl-adenylate (LH2-AMP)22–24, a mixed anhydride. The phosphoryl group transfer is also found, for example, in the activation of fatty acids by fatty acyl-coenzyme A synthetases and in the attachment of amino acids to their correspondent tRNA in protein synthesis by aminoacyl-tRNA synthetases21, 25. Formation of ATP-Mg2+ complexes partially shields the negative charges and influences the conformation of the phosphate groups, and thus explains the requirement of this divalent cation in the reaction26.

The L-LH2 isomer can also be adenylylated, giving rise to L-LH2-AMP; however it is not used further in light production24, 27.

The attachment of a good leaving group like AMP to a metabolic intermediate activates it for subsequent reaction, in this case the oxidation and decarboxylation of the formed D-LH2-AMP [Eq. (2)] [Figs. 5 and 6;21, 28–30].

  • equation image(2)
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Figure 5. Mechanism of dioxetanone formation from D-LH2-AMP. After its formation the intermediate D-LH2-AMP looses a proton to generate a reactive carbanion, whose nucleophilic attack of molecular oxygen creates a hydroperoxide. Internal nucleophilic attack of the hydroperoxide to the electrophilic carbon of the carbonyl group displaces AMP as a leaving group and produces the cyclic dioxetatone ring, an energy-rich moiety.

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Figure 6. Oxidative decarboxylation of the dioxetanone ring in D-LH2 to yield the light-emitter oxyluciferin. The spontaneous break-up of the dioxetanone ring, through a process not yet fully understood, produces oxyluciferin in a singlet excited state (represented by the *) whose decay to the ground level results in the emission of a photon of visible light. The keto and enol tautomers of oxyluciferin are represented in square brackets.

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After the formation of D-LH2-AMP the C4 carbon looses a proton, creating a carbanion. This mechanism explains the function of the adenylylation step: ATP would not serve as an energy donor23 but instead increases the acidity of the C4 carbon30, 31. The carbanion undergoes a nucleophilic attack of molecular oxygen, resulting in a linear hydroperoxide30, 32. The displacement of AMP favors the intramolecular nucleophilic attack of the α-hydroperoxy group towards the formation of a strained four-membered cyclic intermediate, the luciferin dioxetanone33, an energy-rich moiety that spontaneously breaks up generating CO2. Double-labelling experiments with 18O2 and H218O revealed the origin of the oxygen atoms in CO2: in a H216O medium with 18O2 the major portion of the CO2 produced (up to 75%) contained one atom of 18O, which suggests that one of the oxygen of CO2 arises from the O2 that oxidizes LH2, and not from the solvent, thus supporting the dioxetanone mechanism [Fig. 6;34]. The resulting molecule, oxyluciferin, is in a singlet excited state, and its decay to the ground state releases a photon35–37.

Besides the light-producing pathway luciferase catalyzed bioluminescence also displays lateral reactions. In such reaction the complex luciferase·D-LH2-AMP reacts with oxygen in a dark reaction pathway leading to the oxidized product dehydroluciferyl-AMP (L-AMP) [Eq. (3)]23, 38–40.

  • equation image(3)

If the formation of hydrogen peroxide was a natural consequence of the oxidative process in vitro, and that was in fact already demonstrated41, such metabolite could impair biological functions if its concentration is different from the cellular demand. However the existence of luciferase in fireflies' peroxisomes5, an organelle rich in the oxygen detoxifying enzyme catalase, could represent a proof that this reaction is not impossible in vivo41.

By its turn L-AMP is capable of reacting with the PPi-Mg2+ released in the light-producing pathway forming dehydroluciferin (L) and regenerating ATP [Eq. (4)] [Fig. 7;23, 38].

  • equation image(4)
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Figure 7. Pyrophosphorolysis of dehydroluciferyl-adenylate (L-AMP). In this reaction the nucleophilic oxygen at PPi attacks the electrophilic phosphorus at the adenylate moiety of L-AMP, displacing ATP and creating dehydroluciferin (L).

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As L is an oxidative product of LH2 and ATP, through the oxidation of LH2-AMP to L-AMP and its posterior pyrophosphorolysis23, 38–40, it was originally regarded as the light emitter and equivocally called oxyluciferin19, 20, 23, 38. Its chemical synthesis was achieved18 and its role on the bioluminescent reaction, as well as of its adenylated analog L-AMP, has been clarified. The incubation of chemically synthesized L with luciferase and ATP-Mg2+ prior to the addition of LH2 led to inhibition of the light production39. This effect could be exerted not directly by L but instead through its adenylylation to L-AMP. L-AMP is a very strong luciferase inhibitor20, 23, 39, 42, 43 and its synthesis from LH2-AMP can account for about 20% of the LH2 consumed40, 41.

Besides L-AMP the other product of the luciferase-catalyzed reactions that shows marked inhibitory effect is oxyluciferin. These two products are the main responsible for the typical flash pattern of the bioluminescent emitted light. Oxyluciferin is a competitive inhibitor of luciferase (Ki = 0.50 ± 0.03 μM) while L-AMP act as a tight-binding competitive inhibitor (Ki = 3.8 ± 0.7 nM)44.

The formation of L from L-AMP and PPi, although being a lateral reaction, brings benefits to the light-production pathway: a strong inhibitor, L-AMP, is substituted for a less powerful inhibitor, L, which can be more easily removed from luciferase's active site, thus liberating the enzyme for another cycle of reaction23, 42, 45. It is also curious to verify that PPi can be simultaneously an inhibitor of the light reaction (as a product of reaction) and an enhancer at low concentrations (as it removes the strong inhibitor L-AMP through its pyrophosphorolysis to L and ATP)11, 42.

Luciferase as an Acyl-CoA Ligase: Roles of CoA and NTP

The influence of coenzyme A (CoA) in luciferase light-emitting reaction is a well-established phenomenon38: when added to the reaction mixture in the beginning it could stabilize the light emitted, avoiding the characteristic flash profile. Also, when added to a mixture that already produced light, it could induce a secondary flash of light38. But its role on the bioluminescent reaction was not well understood since CoA is not an original reagent in the light reaction. It was demonstrated that CoA is in fact a substrate, reacting with L-AMP and producing dehydroluciferyl-CoA (L-CoA) in a luciferase catalyzed reaction [Eq. (5)]38, 39, 43.

  • equation image(5)

In this scheme the role of CoA could be better explained: by reacting with the strong inhibitor L-AMP another product is formed, L-CoA. This new product is a less powerful inhibitor, so luciferase can continue catalyzing the bioluminescent reaction43, 45.

The chemical synthesis of L-CoA, from L and CoA in the presence of carbonyldiimidazole bypassing the adenylylation step of L to L-AMP, allowed its chemical characterization and confirmed its identity as a product of luciferase catalyzed thiolysis of L-AMP45. Also, it was demonstrated the reversibility of the reaction, through the formation of L-AMP from L-CoA and AMP and posterior pyrophosphorolysis of L-AMP to give L45.

Besides L-AMP, CoA also reacts with L-LH2-AMP24, 46, leading to L-luciferyl-coenzyme A (L-LH2-CoA) [Eq. (6)]. As L-LH2, and hence its adenylated form L-LH2-AMP, functions as an inhibitor of light production46, 47, its reaction with CoA promotes the light-producing pathway.

  • equation image(6)

But how luciferase, a monooxygenase, could react with CoA? A first highlight to this question came from the observation of the resemblance of the biochemistry of the reactions catalyzed by luciferase and some ligases, like fatty acyl-CoA synthetases25. These enzymes catalyze the adenylylation of fatty acids thought its carboxylic acid moiety, with release of PPi, and posterior thioesterification with CoA, in a mechanism very similar to the adenylylation step of luciferase bioluminescent reaction [Eqs. (7) and (8)] (Fig. 8).

  • equation image(7)
  • equation image(8)
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Figure 8. Mechanism of acyl-adenylate formation. In this reaction, catalyzed by synthetases and luciferase, after the adenylylation of the substrate the carbonyl carbon is attacked by the nucleophilic sulphur in the thiol group of coenzyme A (CoA), displacing AMP and creating an acyl-CoA intermediate. R denotes any other part of the substrate not involved in the reaction described.

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After the production of an adenylated intermediate the thiol group of CoA carries out a nucleophilic attack on the mixed anhydride, displacing AMP and forming the thioester acyl-CoA. The question if luciferase is really a descendent of a primitive fatty acyl-CoA synthetase still remains open to discussion, but it was demonstrated that luciferase, in the presence of fatty acids, ATP-Mg2+ and CoA, can catalyze the formation of fatty acyl-CoAs48. The structural similarities between LH2 and arachidonic acid, one of the fatty acid tested as substrate48, could account for luciferase reconnaissance and reaction [Fig. 9;15]. As fatty acids behave as inhibitors of luciferase reaction49 their conversion to fatty acyl-adenylates represents another way of favoring the light production.

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Figure 9. Comparison of the chemical structures of (A) firefly D-LH2 and (B) arachidonic acid, a fatty acid. In (B) arachidonic acid is in its preferred (most stable) conformation, thus highlighting the spatial resemblance between it and D-LH2. Figure adapted from ref.15.

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Besides the demonstration of the ligase-like activity of luciferase, the inverse reaction was also verified: the luciferase-like activity of ligases50. In this study, using larval Tenebrio molitor, a non-luminescent beetle distantly related to fireflies, it was shown that the larvae shines red when injected with LH2, with kinetics parameters similar to those of firefly luciferase, and its fat body extracts produce luminescence in vitro50. However, in a recent study in which these luciferase-like ligase genes were cloned and produced in a functional form, the above results were not confirmed: no light was produced in vitro by the addition of D-LH2 and ATP-Mg2+51.

Another consequence of luciferase's function as a ligase is the synthesis of dinucleoside polyphosphates, in a reaction analogous to the pyrophosphorolysis of L-AMP to give ATP [Fig. 7;40, 52]. The synthesis of these “mysterious” compounds, found in numerous organisms but without a clear biological function53, was demonstrated to be catalyzed by luciferase, like does acyl-CoA synthetase from Pseudomonas fragi54, from L-AMP and any substance with an intact terminal phosphate, like nucleoside 5′-triphosphates (NTPs, for example CTP, UTP and ATP) and tripolyphosphate (P3), as acceptors of the AMP moiety [Eqs (9)(11)]40, 42, 55, giving adenosine (5′)tetraphospho(5′)nucleoside (Ap4N) or adenosine 5′-tetraphosphate (p4A) as products, respectively. The formation of adenosine(5′)tetraphospho(5′)adenosine (Ap4A) from ATP could account for the consumption of the ATP diverged from the bioluminescent reaction. Nonetheless the best conditions for the synthesis of dinucleoside polyphosphates are the opposite of those necessary for light production, namely acidic medium, while luciferase requires a physiological pH, about 7.5, to produce light55. This contradictory effect was explained as the low pH could facilitate the transfer of the adenylate moiety of L-AMP to the acceptor55.

  • equation image(9)
  • equation image(10)
  • equation image(11)

Once again L is produced, explaining the slightly activator effect verified for the referred acceptors39, 42.

Figure 10 resumes the reactions enumerated above and shows the interconnections between them.

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Figure 10. Schematic representation of luciferase catalyzed reactions. The bioluminescent reaction involves the formation of D-LH2-AMP from D-LH2 and ATP-Mg2+, with the release of PPi, and its posterior oxidative decarboxylation to oxyluciferin, the light emitter, releasing AMP, CO2 and a photon. Lateral (dark) reactions includes the oxidation of D-LH2-AMP to L-AMP, generating hydrogen peroxide (H2O2), and its reaction with PPi and/or a nucleoside 5′-triphosphate (NTP), leading to ATP and adenosine (5′)tetraphospho(5′)nucleoside (Ap4N), respectively, and L. L-AMP and L-LH2-AMP can react with CoA, generating AMP and the products dehydroluciferyl-coenzyme A (L-CoA) and L-luciferyl-coenzyme A (L-LH2-CoA). Figure adapted from ref.24.

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FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

Although the chemical structure and properties of LH2 are already established little is known about its biosynthesis15. As already mentioned, the in vitro (chemical) synthesis of LH2 leads to two enantiomers, D- and L-, according to the cysteine isomer used. In Nature only the L-form of amino acids occurs in peptides and proteins, therefore it has been speculated where does the D-cysteine for the synthesis of D-LH2 comes from.

It was suggested that D-LH2 could be produced from L-LH2 by an enzyme-catalyzed inversion of the latter with participation of CoA [Fig. 11;56]. According to this hypothesis, L-LH2 is produced from natural L-cysteine, and then L-LH2 is converted into L-LH2-CoA which is racemized by enolization56. Hydrolysis of D-LH2-CoA would give the bioluminescent substrate, D-LH2. The production of LH2-CoA is stereospecific46, resulting only from L-LH2, which indicates that luciferase is able to distinguish the two isomers of LH2, serving as the acyl-CoA synthetase for L-LH2, and D-LH2 being used for the bioluminescent reaction46. This pathway could explain another characteristic of luciferase bioluminescence: the production of light from L-LH247.

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Figure 11. Proposed biosynthetic pathway of D-LH2 from L-LH2. Beginning with 2-cyano-6-hydroxybenzothiazole (CHBT) and the natural L-cysteine the L-LH2 isomer is formed. Through an intermediary step of adenylylation this isomer is converted into L-LH2-AMP, and its posterior reaction with CoA generates L-LH2-CoA, being both reactions catalyzed by luciferase. The keto-enol equilibrium leads to racemization of the isomers L- and D-LH2-CoA which are then hydrolyzed to L-LH2 and D-LH2, the latter being the bioluminescent substrate. Figure adapted from ref.56.

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For a long time D-LH2 was regarded as the only isomer capable of producing light14, 27, the L-LH2 isomer behaving as an inhibitor46, 47, 57, although both D- and L- isomer can be adenylylated by luciferase. Through a mechanism analogous to its synthesis the added L-LH2 would be adenylylated, converted to L-LH2-CoA and then racemized and hydrolyzed to D-LH2. However, the light profile is quite different from the one with directly added D-LH2, namely presenting a slow increase in the light emission until reaching a plateau, and weaker luminescence intensity47.

Besides the de novo synthesis of LH2 another hypothesis is its regeneration from oxyluciferin, proposed on the basis of the discovery of the luciferin-regenerating enzyme (LRE) in Photinus pyralis [Fig. 12;58]. LRE convertes oxyluciferin to 2-cyano-6-hydroxybenzothiazole (CHBT) and thioglycolic acid. In the presence of D-cystein CHBT was recycled into D-LH2 by a non-enzymatic reaction. In the same study this activity was detected in extracts from Luciola cruciata and Luciola lateralis, but further research on this subject is needed58.

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Figure 12. Recycling pathway for LH2 catalyzed by luciferin-regenerating enzyme (LRE). After the bioluminescent reaction catalyzed by luciferase the natural substrate D-LH2 is transformed into oxyluciferin. LRE then converts oxyluciferin into 2-cyano-6-hydroxybenzothiazole (CHBT), with the formation of thioglycolic acid as a co-product. In a putative non-catalyzed reaction the condensation of D-cysteine with CHBT produces D-LH2 that can enter the next cycle of light emission. Figure adapted from ref.58.

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APPLICATIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

The studies aimed to the comprehension and characterization of the bioluminescent reaction allowed the development of luciferase-based techniques, relying in the intrinsic characteristics of enzymatic assays, specificity and sensitivity, and, in the particular case of luciferase, in the possibility of light measurement. In general there are two broad areas of interest: the utilization of luc gene as a reporter in molecular biology studies and bioimaging59, or the quantification of analytes connected to ATP or other participant of the light reaction.

The isolation and purification of luciferases from different species was the first step into their practical applications, and today commercial kits are available. The most popular analyte quantified by luciferase is ATP, due to the early discovery that the light intensity is proportional to the ATP concentration in the sample analyzed2 and to the vital importance of this compound to life. On the basis of Eqs. (1) and (2) a simple system can be composed of luciferase, D-LH2 and the sample to be quantified. Sometimes CoA is also added to the reaction mixture to stabilize the light emitted. ATP quantitation is a fundamental procedure in food industries, as an indicator of bacterial contamination60.

Besides ATP the attention was also turned to other biologically important metabolites which participates in luciferase-catalyzed reaction, namely CoA [through Eq. (5)]61, PPi62, 63 and AMP64, 65.

Recently the discovery that L-LH2 can also be used by luciferase to produce light led to the development of a new system which involves L-LH2, ATP, CoA, luciferase and a esterase and was applied to monitoring luciferase concentration itself66. In fact, the use of luciferase to monitor enzyme activity was already applied for pyrophosphatase (PPase)67. The addition of PPi to the assay medium inhibits luciferase, but PPase degrades PPi and thus restores luciferase activity, being the light output proportional to the PPase content67.

Albeit purified luciferase is an important and well established analytical tool it was necessary to wait until recently for progresses in molecular biology that allowed the cloning and sequencing of luciferases genes68. But soon after that a new application for luciferases was developed as a reporter gene. A reporter gene is the one which generates a measurable signal according to certain conditions, reporting about cellular functions like the pattern of gene expression, cellular receptors activity, signal transduction pathway, RNA processing and protein–protein interaction69. The luc gene is introduced into the desired organism by means of an expression vector, like a plasmid. Inside the cell the luc gene is translated into functional luciferase protein. An extract is made and the addition of D-LH2 and ATP-Mg2+ leads to light emission that can be recorded in a luminometer (Fig. 13). This strategy was applied to identify regulatory sequences or promoter regions in the genome70, 71 and in drug screening72–74.

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Figure 13. Schematic representation of the reporter gene technology related to luciferase. An expression vector is introduced into the cell. The vector contains a regulatory sequence (A), which will act upon the luc gene (B), controlling its expression. When the expression of luc is promoted the protein obtained (Luciferase) can be assayed by its light producing activity in vitro (cellular extracts) or in vivo, placing the animal under a charge-coupled device (CCD) camera that is sensible to detect photons, enabling a luminous spot of light that can be artificially superimposed with a photo of the animal previously taken. In both cases ATP and D-LH2 must be exogenously administered just before data acquisition. Figure adapted from ref.59 and text from refs.59 and79.

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On the basis of this principle another technique was developed: the bioimaging, in which the light production is followed in vivo in a whole organism (Fig. 13). This area is receiving much attention and it is applied in important fields like oncology (to evaluate tumor growth and metastases formation)75, 76, infection progression74, 77, and protein expression under specific stimulus78. The principal drawback is that this technique is not yet applied in clinical studies: the actual investigation relies only in animal model79.

Finally, mutagenesis studies on luciferase enabled the production of proteins with new and enhanced properties, like new light color emission80 improved thermostability81, higher luminescence intensity82 and increased catalytic efficiency83, 84.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

As a complex system that it is, the knowledge about luciferase's bioluminescence is still being built up. Albeit much progress was done during the past decades, many mysteries remain unsolved. In fact even the mechanism of luciferase's action in all reactions shown in this paper is not completely clear until now. Questions as “Which amino acid residues take part in each catalytic step of the reaction?” or “What intermediate enzyme-substrate states are formed during reaction?” are examples of problems that should be elucidated in firefly bioluminescent catalysis. Nonetheless, novel applications based on luciferase are currently being developed and new discoveries are likely to occur in the near future. The unification of so diverse fields as molecular biology, genetics and chemistry will certainly bring to light a new era of bioluminescence research.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES

A research grant (to Simone M. Marques) from the Faculdade de Ciências da Universidade do Porto is acknowledged. Financial support from Universidade do Porto and Caixa Geral de Depósitos (Project IPG136) and from Fundação para a Ciência e Tecnologia (Lisboa) (FSE-FEDER) (Project POCTI/QUI/37768/2001) is also acknowledged.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FIREFLY LUCIFERASE
  5. FIREFLY LUCIFERASE REACTIONS
  6. FIREFLY LUCIFERIN BIOSYNTHESIS AND THE STEREOSPECIFICITY IN FIREFLY BIOLUMINESCENCE
  7. APPLICATIONS
  8. CONCLUSION
  9. Acknowledgements
  10. REFERENCES