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The phenomenon of bioluminescence has been observed in many different organisms including bacteria, fungi, fish, squid, algae, shrimp and insects. Luminous organisms produce light by an enzymatic reaction of a luciferase (=enzyme) with a luciferin (=substrate). Light-emitting reactions are quite distinct among luminous organisms, but in each case the reaction is an oxidation process with molecular oxygen and is a conversion of chemical energy into light. The various luciferase genes have been isolated and adapted as reporter genes. In insects, the luminous species are mainly found in three families: fireflies (Lampyridae), click beetles (Elateridae) and railroad worms (Phengodidae). The bioluminescence systems of these insects are essentially the same with an identical luciferin, ATP, and Mg2+, and with similar luciferases. Firefly luciferase (FLuc) has been studied for the last 50 years and proven to be a useful enzyme. It has been extensively used in molecular and cell biology, in particular for the quantification of ATP and as a reporter enzyme of gene expression.
Overview of firefly luciferases
One of the main reasons for the widespread use of luciferase in bioassays is that, unlike fluorescence, luminescence does not require excitation light energy, so lowering the background signal to provide a sensitive assay with a high signal-to-background ratio. Elimination of an excitation light source also prevents interference by compound fluorescence and fluorophore photobleaching. Therefore, luminescence assays can be very sensitive, despite significantly weaker signal intensity than fluorescence. For cell-based assays, the use of FLuc or Renilla luciferase (RLuc) enzymes as reporters enable the measurement of dynamic changes in reporter transcription levels because the intracellular protein half-lives of these luciferase enzymes are relatively short compared to nonenzymatic fluorescent protein reporters (e.g., GFP).
The application of FLuc as a reporter began in the late 1980s, following its initial cloning, and expression in mammalian cells. The FLuc enzyme was found to be a sensitive reporter for biological assays and catalytically active as a monomer and was optimized for expression and activity as a reporter gene in mammalian cells, though it is now widely used for biochemical applications in high-throughput screening (HTS) as well. A luciferase from a different species of firefly, Photuris pennsylvanica, has also been optimized and developed for use as a reporter on the basis of its utilization of D-luciferin (D-LH2) and ATP as substrates (e.g., Ultra-Glo). Typical biochemical assays that utilize either FLuc or Ultra-Glo include those that measure ATP or ADP concentrations or use pro-luciferin substrates to detect target protein activity.
Structure and function of FLuc
FLuc is a member of a superfamily of adenylate-forming enzymes, which include acyl- and aryl-CoA synthetases, and nonribosomal peptide synthetases and by primary sequence homology, appears to be most closely related to certain acyl-CoA ligases. Transfer of an adenylate group to a substrate to form an enzyme-associated intermediate is, mechanistically, a common mode of substrate activation in this enzyme family.
FLuc is an ATP-hydrolyzing, decarboxylating 4-oxidoreductase that requires a luciferin substrate containing a hydroxy-benzothiazolyl-thiazoline-carboxylic acid structure (D-LH2), oxygen, ATP and a metal cation (typically Mg2+) to produce light. Bioluminescence is produced from D-LH2 by FLuc through an SN2 nucleophilic displacement reaction in which the carboxylate on the thiazoline ring attacks the a-phosphoryl moiety of ATP. The first step in the reaction results in the release of PPi and the formation of an enzyme-bound luciferyl-adenylate, LH2-AMP. The second step of the reaction involves the oxidation of LH2-AMP by molecular oxygen. This leads to the production of an unstable luciferin dioxetanone, which generates CO2 and an electronically excited oxyluciferin which, upon spontaneous decay to the ground state, generates photons of both red and green light (Figure 1).
Figure 1. Mechanism of the bioluminescence reaction of firefly luciferin by FLuc.
Dual-luciferase assay system
The luciferases FLuc, RLuc, and the red- or green-emitting Caribbean click beetle (CBLuc) varieties have been configured for use in dual-luciferase assays. One such dual-luciferase assay is designed such that activity from one luciferase (typically FLuc) tracks with the target biology while the second luciferase (e.g., RLuc) is used for assay normalization (to account for cytotoxicity, differences in cell number, and transfection efficiency in the case of transiently transfected cell lines). In this dual-luciferase assay system, measurement of the two luciferases is sequential, requiring separate substrate addition steps (Figure 2), because the two luciferases need different substrates and collection of the second luminescence signal requires termination of the preceding reaction. In the case of disparate enzymes, such as FLuc and RLuc, which are evolutionarily unrelated and have different substrate specificities, it is expected that these two enzymes will have dissimilar inhibition profiles. Therefore, the changes in the FLuc/RLuc luminescence ratio intended to be reflective of target modulation could actually be the result of the direct and selective inhibition of either luciferase reporter enzyme.
Figure 2. The dual luciferase assay protocol.