| Luciferase-like monooxygenase | |||||||||
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| Structure of the non-fluorescent flavoprotein from Photobacterium leiognathi.[1] | |||||||||
| Identifiers | |||||||||
| Symbol | Bac_luciferase | ||||||||
| Pfam | PF00296 | ||||||||
| InterPro | IPR016048 | ||||||||
| PROSITE | PDOC00397 | ||||||||
| SCOP | 1nfp | ||||||||
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| Firefly luciferase | |
|---|---|
| Structure of Photinus pyralis firefly luciferase. | |
| Identifiers | |
| Symbol | Firefly luciferase |
| PDB | 1LCI |
| UniProt | P08659 |
| Other data | |
| EC number | 1.13.12.7 |
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis.[2] "Firefly luciferase" as a laboratory reagent usually refers to P. pyralis luciferase although recombinant luciferases from several other species of fireflies are also commercially available. The name is derived from Lucifer, the root of which means 'light-bearer' (lucem ferre).
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In luminescent reactions, light is produced by the oxidation of a luciferin (a pigment):
The most common luminescent reactions release CO2 as a product. The rates of this reaction between luciferin and oxygen are extremely slow until they are catalyzed by luciferase, sometimes mediated by the presence of cofactors such as calcium ions or ATP.[3] The reaction catalyzed by firefly luciferase takes place in two steps:
Nearly all of the energy input into the reaction is transformed into light. The reaction is 80%[4] to 90%[5] efficient. As a comparison, the incandescent light bulb only converts about 10% of its energy into light.[6] and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.[5]
A subtle structural difference in luciferase has been discovered to be the cause of the change in bioluminescence emission colour from a yellow-green to red. The structure of wild-type luciferase and red mutant (S286N) luciferase from the Japanese Genji-Botaru (Luciola cruciata) in complex with an intermediate analogue 5’-O-[N-(dehydroluciferyl)-sulfamoyl] adenosine (DLSA) was examined and studies showed that the wild-type luciferase complexed with DLSA exhibited a ‘closed form’ of the active site, where the side chain of amino acid isoleucine 288 moved towards the benzothiazole ring of DLSA, creating a rigid hydrophobic pocket. The ‘closed form’ wild-type luciferase bound the excited state of oxyluciferin in a highly rigid and nonpolar microenvironment, minimizing energy loss before emitting yellow-green light. The S286N luciferase complexed with DLSA exhibited an ‘open form’ of the active site, where the amino acid side chain of isoleucine 288 did not move towards the benzothiazole ring of DLSA, creating a less rigid and less hydrophobic microenvironment. The ‘open form’ S286N luciferase had a less rigid microenvironment allowing some energy loss from the excited state of oxyluciferin, which resulted in the emission of low-energy red light.[7]
A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the fireflies,[8] although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea. The luciferases of fireflies - of which there are over 2000 species - and of the Elateroidea (fireflies, click beetles and relatives) in general - are diverse enough to be useful in molecular phylogeny. The most thoroughly studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8.[9]
Also well studied is the luciferase from Renilla reniformis. In this organism, the luciferase is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:[10]
Newer luciferases have recently been identified that, unlike Renilla or Firefly luciferase, are naturally secreted molecules. One such example is the Metridia luciferase (MetLuc)that is derived from the marine copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.[11]
Luciferase can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. Mice, silkworms, and potatoes are just a few organisms that have already been engineered to produce the protein.[12]
In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes.[13]
In biological research, luciferase commonly is used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest.[14] Luciferase can also be used to detect the level of cellular ATP in cell viability assays or for kinase activity assays.[14][15] Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase activity and cytochrome P450 activity, among others.[13][14]
Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice.[16] Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive charge-couple device camera (CCD camera).This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models.[17][18] However, environmental factors and therapeutic interferences may cause some discrepancies between tumor burden and bioluminescence intensity in relation to changes in proliferative activity. The intensity of the signal measured by in vivo imaging may depend on various factors, such as D-luciferin absorption through the peritoneum, blood flow, cell membrane permeability, availability of co-factors, intracellular pH and transparency of overlying tissue, in addition to the amount of luciferase.[19]
Luciferase can be used in blood banks to determine if red blood cells are starting to break down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for using luciferase continue to expand.[20]
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