Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered.

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc.

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Abstract— Twenty‐five Brazilian species (nine genera: Phorinus, Photinoides, Macrolampis, Aspisoma, Cratomorphus, Amydetes, Photuris, Bicellonychia, Pyrogaster) of adult fireflies were found to emit light in vivo in the green‐yellow range (Λmax=548–573 nm) of the spectrum, more frequently near the green region, in contrast with North‐American species, which predominantly emit yellow light. Distinct ecological contexts where these species evolved, such as the habitat (open field vs forests) and the duration of twilight, are discussed as possible factors responsible for these differences. Except for Photuris and Bicellonychia spp., the in vivo and in vitro bioluminescence spectra for various species of a given genus agree within ±5 nm. Lowering the pH caused the typical red shift in the in vitro bioluminescence spectrum from lampyrid luciferases (six species), which has been interpreted as due to the presence of a basic residue in the enzyme active site catalyzing fast enolization of the initially formed excited keto‐oxyluciferin (red emitter) to the excited enol form (yellow‐green emitter). The in vitro bioluminescence colors obtained from larval or adult elaterid (five species) and phengodid (three species) luciferases studied here, spanning the green‐red region, do not respond to pH changes. This could indicate either the absence of the neighboring basic center (in red‐emitting luciferases) or the presence of a non‐pH affected proximal basic residue in the active site of the luciferase (in yellow‐green‐emitting luciferases).

The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil. The bioluminescence (BL) spectrum of this larva (lambda max = 534 nm) is one of the most blue-shifted reported among known luminescent Coleoptera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (lambda max = 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.

Among beetle luciferases, the pH-sensitive firefly luciferases have been studied extensively. Much less is known about pH-insensitive luciferases, which include click beetle and railroad worm luciferases. Previously, we found that the residues R215 and T226 (N230) are important for green light emission. Here we show that the conserved residue A243 in pH-insensitive luciferases and the corresponding G247 in pH-sensitive luciferases affect the emission spectrum and influence pH-sensitivity. In contrast to railroad worm green light-emitting (PxvGR) and firefly luciferases, the substitution of R215 in Pyrearinus termitilluminans click beetle luciferase (Pte) had no effect on the spectrum, showing that R215 is not essential for green light emission in all beetle luciferases. A homology-based model of Pte luciferase shows that R215 and T226 are close enough to interact. To investigate if there was an interaction between these conserved residues, double mutants were constructed. The double substitution R215S/T226N in Pte luciferase abolished the activity. In PxvGR luciferase the same double mutant resulted in a redshift (lambda(max) = 595 nm), whose magnitude was lower than the value expected for an additive effect. These results suggest that the effects of R215S and T226N are partially interdependent. The double substitution T226N/A243G had an additive redshift effect on the spectrum of PxvGR luciferase, whereas it had a smaller effect on the spectrum of Pte luciferase. Altogether, these results suggest that the above substitutions have different effects on the active site of click beetle and railroad worm luciferases.