| Bacterial Luciferase monooxygenase family | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||||
| Symbol | Bac_luciferase | ||||||||||
| Pfam | PF00296 | ||||||||||
| InterPro | IPR016048 | ||||||||||
| PROSITE | PDOC00397 | ||||||||||
| SCOP2 | 1nfp /SCOPe /SUPFAM | ||||||||||
| |||||||||||
| Dinoflagellate Luciferase catalytic domain | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 crystal structure of a luciferase domain from the dinoflagellateLingulodinium polyedrum | |||||||||
| Identifiers | |||||||||
| Symbol | Luciferase_cat | ||||||||
| Pfam | PF10285 | ||||||||
| InterPro | IPR018804 | ||||||||
| |||||||||
| Dinoflagellate Luciferase/LBP N-terminal domain | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| Symbol | Luciferase_N | ||||||||
| Pfam | PF05295 | ||||||||
| InterPro | IPR007959 | ||||||||
| |||||||||
| Dinoflagellate Luciferase helical bundle domain | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| Symbol | Luciferase_3H | ||||||||
| Pfam | PF10284 | ||||||||
| InterPro | IPR018475 | ||||||||
| |||||||||
| Firefly luciferase | |||||||
|---|---|---|---|---|---|---|---|
 Structure ofPhotinus pyralisfirefly luciferase. | |||||||
| Identifiers | |||||||
| Organism | Photinus pyralis | ||||||
| Symbol | Firefly luciferase | ||||||
| PDB | 1LCIMore structures | ||||||
| UniProt | P08659 | ||||||
| Other data | |||||||
| EC number | 1.13.12.7 | ||||||
| |||||||
Luciferase is a generic term for the class of oxidativeenzymes that producebioluminescence, and is usually distinguished from aphotoprotein. The name was first used byRaphaël Dubois who invented the wordsluciferin andluciferase, for the substrate andenzyme, respectively.[1] Both words are derived from the Latin wordlucifer, meaning "lightbearer", which in turn is derived from the Latin words for "light" (lux) and "to bring or carry" (ferre).[2]Luciferases are widely used inbiotechnology, forbioluminescence imaging[3] microscopy and asreporter genes, for many of the same applications asfluorescent proteins. However, unlike fluorescent proteins, luciferases do not require an externallight source, but do require addition ofluciferin, the consumable substrate.
A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The majority of studied luciferases have been found in animals, includingfireflies,[4] and many marine animals such ascopepods,jellyfish, and thesea pansy. However, luciferases have been studied in luminous fungi, like theJack-O-Lantern mushroom, as well as examples in other kingdoms includingbioluminescent bacteria, anddinoflagellates.
Theluciferases of fireflies – of which there are over 2000species – and of the otherElateroidea (click beetles and relatives in general) are diverse enough to be useful inmolecular phylogeny.[5] In fireflies, the oxygen required is supplied through a tube in the abdomen called theabdominaltrachea. One well-studied luciferase is that of thePhotinini fireflyPhotinus pyralis, which has an optimum pH of 7.8.[6]
Also well studied is the sea pansy,Renilla reniformis. In this organism, the luciferase (Renilla-luciferin 2-monooxygenase) 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 foroxidation 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 throughresonance energy transfer to thefluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:[8]
Newer luciferases have recently been identified that, unlike other luciferases, are naturally secreted molecules. One such example is theMetridiacoelenterazine-dependent luciferase (MetLuc,A0A1L6CBM1) that is derived from the marine copepodMetridia longa. TheMetridia longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signalpeptide of 17amino 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.[9]
Bacterial bioluminescence is seen in Photobacterium species,Vibrio fischeri andVibrio harveyi. Light emission in somebioluminescent bacteria utilizes 'antenna' such aslumazine protein to accept the energy from the primary excited state on the luciferase, resulting in an excitedlulnazinechromophore which emits light that is of a shorter wavelength (more blue), while in others use ayellow fluorescent protein (YFP) withflavin mononucleotide (FMN) as the chromophore and emits light that is red-shifted relative to that from luciferase.[10]
Dinoflagellate luciferase is a multi-domaineukaryote protein, consisting of anN-terminal domain, and threecatalytic domains, each of which preceded by a helical bundle domain. Thestructure of the dinoflagellate luciferasecatalyticdomain has been solved.[11] The core part of the domain is a 10 strandedbeta barrel that isstructurally similar tolipocalins andFABP.[11] The N-terminal domain isconserved between dinoflagellate luciferase andluciferin binding proteins (LBPs). It has been suggested that this region may mediate an interaction between LBP and luciferase or their association with thevacuolar membrane.[12]The helical bundle domain has a threehelix bundlestructure that holds four importanthistidines that are thought to play a role in thepH regulation of theenzyme.[11] There is a large pocket in the β-barrel of the dinoflagellate luciferase at pH 8 to accommodate thetetrapyrrole substrate but there is no opening to allow the substrate to enter. Therefore, a significant conformational change must occur to provide access and space for aligand in the active site and the source for this change is through the four N-terminal histidine residues.[11] At pH 8, it can be seen that the unprotonated histidine residues are involved in a network ofhydrogen bonds at the interface of the helices in the bundle that block substrate access to theactive site and disruption of this interaction byprotonation (at pH 6.3) or by replacement of the histidine residues byalanine causes a large molecular motion of the bundle, separating the helices by 11Å and opening the catalytic site.[11] Logically, the histidine residues cannot be replaced by alanine in nature but this experimental replacement further confirms that the larger histidine residues block the active site. Additionally, three Gly-Gly sequences, one in the N-terminal helix and two in the helix-loop-helix motif, could serve as hinges about which the chains rotate in order to further open the pathway to the catalytic site and enlarge the active site.[11]
A dinoflagellate luciferase is capable of emitting light due to its interaction with its substrate (luciferin) and the luciferin-binding protein (LBP) in thescintillonorganelle found in dinoflagellates.[11] The luciferase acts in accordance with luciferin and LBP in order to emit light but each component functions at a different pH. Luciferase and its domains are not active at pH 8 but they are extremely active at the optimum pH of 6.3 whereas LBP binds luciferin at pH 8 and releases it at pH 6.3.[11] Consequently, luciferin is only released to react with an active luciferase when the scintillon is acidified to pH 6.3. Therefore, in order to lower the pH,voltage-gated channels in the scintillonmembrane are opened to allow the entry ofprotons from avacuole possessing anaction potential produced from a mechanical stimulation.[11] Hence, it can be seen that the action potential in the vacuolar membrane leads to acidification and this in turn allows the luciferin to be released to react with luciferase in the scintillon, producing a flash of blue light.
All luciferases are classified asoxidoreductases (EC1.13.12.-), meaning they act onsingle donors with incorporation ofmolecular oxygen. Because luciferases are from many diverseprotein families that are unrelated, there is no unifying mechanism, as any mechanism depends on the luciferase and luciferin combination. However, all characterised luciferase-luciferin reactions[13] to date have been shown to require molecularoxygen at some stage.
FireflyPhotinus pyralis luciferase in the adenylate-forming conformation bound to DLSA. Key interaction observed between K529 and carbonyl oxygen ofadenylate. PDB 4G36]]
The luciferase ofPhotinus pyralis catalyzes a two-step bioluminescent reaction. First isadenylation, a process in which D-luciferin is converted to D-luciferyl-adenylate (D-AMP) via the covalent addition ofadenosine monophosphate to an amino acid side chain. Next,oxidative decarboxylation of the adenylated intermediate occurs, a necessary step for light emission. Studies have presented the first crystal structure of luciferase in its second catalytic conformation using DLSA (5′-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine), a stable analog of D-AMP. The Photinus pyralis luciferase in the adenylate-forming conformation bound to DLSA illustrates conserved interactions observed in other adenylate-forming enzymes as well as key insights into the mechanism ofbioluminescence. The active site is located at the interface of the N-terminal and C-terminal domains. Lys529 is the catalytic lysine for the initial adenylation reaction, interacting with the carbonyl oxygen of the ligand. A transition to the oxidation-available conformation involves a ~140° rotation of the C-terminal domain, upon which oxidation initiates formation of the dioxetanone intermediate. The decomposition of this intermediate releases visible light. Unlike D-AMP, DLSA cannot undergo oxidation, but the “locking” of the enzyme in its second catalytic conformation allowed researchers to study the oxidation-ready state of luciferase.[14]
| Bacterial Luciferase monooxygenase family | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||||
| Symbol | Bac_luciferase | ||||||||||
| Pfam | PF00296 | ||||||||||
| InterPro | IPR016048 | ||||||||||
| PROSITE | PDOC00397 | ||||||||||
| SCOP2 | 1nfp /SCOPe /SUPFAM | ||||||||||
| |||||||||||
The reaction catalyzed by bacterial luciferase is also an oxidative process:
In the reaction, molecular oxygen oxidizesflavin mononucleotide and a long-chain aliphaticaldehyde to an aliphaticcarboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to the product FMN to emit blue-green light.[15]
Nearly all of the energy input into the reaction is transformed into light. The reaction is 80%[16] to 90%[17] efficient. In comparison, theincandescent light bulb only converts about 10% of itsenergy into light[18] and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.[17]
Luciferases can be produced in the lab throughgenetic engineering for a number of purposes. Luciferasegenes can be synthesized and inserted into organisms ortransfected into cells. As of 2002,mice,silkworms, andpotatoes are just a few of the organisms that have already been engineered to produce the protein.[19]
In the luciferase reaction, light is emitted when luciferase acts on the appropriateluciferinsubstrate. Photon emission can be detected by light sensitive apparatus such as aluminometer or anoptical microscope with aCCD camera. This allows observation of biological processes.[20] Since light excitation is not needed for luciferase bioluminescence, there is minimalautofluorescence and therefore the bioluminescent signal is virtually background-free.[21] Therefore, as little as 0.02 pg can still be accurately measured using a standardscintillation counter.[22]
In biological research, luciferase is commonly used as a reporter to assess thetranscriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of apromoter of interest.[23] 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 detectcaspase activity andcytochrome P450 activity, among others.[20][23]
Luciferase can also be used to detect the level of cellular ATP in cell viabilityassays or for kinase activity assays.[23][24] Luciferase can act as an ATP sensor protein throughbiotinylation. Biotinylation will immobilize luciferase on the cell-surface by binding to astreptavidin-biotin complex. This allows luciferase to detect the efflux of ATP from the cell and will effectively display the real-time release of ATP through bioluminescence.[25] Luciferase can additionally be made more sensitive for ATP detection by increasing the luminescence intensity by changing certainamino acidresidues in the sequence of the protein.[26]
Whole organism imaging (referred to asin vivo when intact or, otherwise calledex vivo imaging for example of living but explanted tissue) is a powerful technique for studying cell populations in live plants or animals, such as mice.[27] 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.[28][29] 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 asD-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.[30]
Luciferase is a heat-sensitive protein that is used in studies onprotein denaturation, testing the protective capacities ofheat shock proteins. The opportunities for using luciferase continue to expand.[31]
Media related toLuciferases at Wikimedia Commons