Luciferase

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Bacterial Luciferase monooxygenase family
Identifiers
SymbolBac_luciferase
SCOP2
1nfp / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDB1brl​, 1bsl​, 1ezw​, 1fvp​, 1luc​, 1m41​, 1nfp​, 1nqk​, 1rhc​, 1xkj​
Dinoflagellate Luciferase catalytic domain
crystal structure of a luciferase domain from the dinoflagellate Lingulodinium polyedrum
Identifiers
SymbolLuciferase_cat
PfamPF10285
InterProIPR018804
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Dinoflagellate Luciferase/LBP N-terminal domain
Identifiers
SymbolLuciferase_N
PfamPF05295
InterProIPR007959
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Dinoflagellate Luciferase helical bundle domain
Identifiers
SymbolLuciferase_3H
PfamPF10284
InterProIPR018475
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Luciferase is a generic term for the class of oxidative

UniProtP08659Other dataEC number1.13.12.7
Search for
StructuresSwiss-model
DomainsInterPro

Luciferases are widely used in biotechnology, for bioluminescence imaging[3] microscopy and as reporter genes, for many of the same applications as fluorescent proteins. However, unlike fluorescent proteins, luciferases do not require an external light source, but do require addition of luciferin

, the consumable substrate.

Examples

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, including

dinoflagellates
.

Firefly and click beetle

The

trachea. One well-studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8.[6]

Sea pansy

Also well studied is the

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:[7]

Copepod

Newer luciferases have recently been identified that, unlike other luciferases, are naturally secreted molecules. One such example is the Metridia coelenterazine-dependent luciferase (MetLuc, A0A1L6CBM1) 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.[8]

Bacterial

Bacterial bioluminescence is seen in Photobacterium species,

Vibrio fischeri, Vibrio haweyi, and Vibrio harveyi. Light emission in some bioluminescent bacteria utilizes 'antenna' such as lumazine protein to accept the energy from the primary excited state on the luciferase, resulting in an excited lulnazine chromophore which emits light that is of a shorter wavelength (more blue), while in others use a yellow fluorescent protein (YFP) with flavin mononucleotide (FMN) as the chromophore and emits light that is red-shifted relative to that from luciferase.[9]

Dinoflagellate

FABP.[10]
The N-terminal domain is
vacuolar membrane.[11]
The helical bundle domain has a three
hydrogen bonds at the interface of the helices in the bundle that block substrate access to the active site and disruption of this interaction by protonation (at pH 6.3) or by replacement of the histidine residues by alanine causes a large molecular motion of the bundle, separating the helices by 11Å and opening the catalytic site.[10] 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.[10]

A dinoflagellate luciferase is capable of emitting light due to its interaction with its substrate (

protons from a vacuole possessing an action potential produced from a mechanical stimulation.[10]
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.

Mechanism of reaction

All luciferases are classified as

oxidoreductases (EC 1.13.12.-), meaning they act on single donors with incorporation of molecular oxygen. Because luciferases are from many diverse protein 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 to date have been shown to require molecular oxygen
at some stage.

Bacterial luciferase

The reaction catalyzed by bacterial luciferase is also an oxidative process:

  • FMNH2 + O2 + RCHO → FMN + RCOOH + H2O + light

In the reaction, molecular oxygen oxidizes flavin mononucleotide and a long-chain aliphatic aldehyde to an aliphatic carboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to the product FMN to emit blue-green light.[12]

Nearly all of the energy input into the reaction is transformed into light. The reaction is 80%

incandescent light bulb only converts about 10% of its energy into light[15] and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.[14]

Applications

Luciferases can be produced in the lab through

silkworms, and potatoes are just a few of the organisms that have already been engineered to produce the protein.[16]

In the luciferase reaction, light is emitted when luciferase acts on the appropriate

CCD camera. This allows observation of biological processes.[17] Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and therefore the bioluminescent signal is virtually background-free.[18] Therefore, as little as 0.02 pg can still be accurately measured using a standard scintillation counter.[19]

In biological research, luciferase is commonly used as a reporter to assess the

promoter of interest.[20] 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.[17][20]

Luciferase can also be used to detect the level of cellular ATP in cell viability assays or for kinase activity assays.[20][21] Luciferase can act as an ATP sensor protein through biotinylation. Biotinylation will immobilize luciferase on the cell-surface by binding to a streptavidin-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.[22] Luciferase can additionally be made more sensitive for ATP detection by increasing the luminescence intensity by changing certain amino acid residues in the sequence of the protein.[23]

Graph with four rhythmic lines that peak in pairs, at alternating times over six, 24-hour cycles.
Graph showing timeseries data from in vivo imaging of firefly luciferase bioluminescence. Transgenic seedlings of Arabidopsis thaliana were imaged by a cooled CCD camera under three cycles of 12h light: 12h dark followed by 3 days of constant light. Their genomes carry firefly luciferase reporter genes, so the signals of seedlings 61 (red) and 62 (blue) reflect transcription of the gene CCA1, while 64 (pale grey) and 65 (teal) reflect TOC1. The timeseries show 24-hour, circadian rhythms of gene expression in the living plants.

Whole organism imaging (referred to as in vivo when intact or, otherwise called ex 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.

CCD camera).This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models.[25][26] 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.[27]

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.[28]

See also

References

  1. ^ Lee J (28 February 2014). "A History of Bioluminescence". photobiology.info. Archived from the original on 29 March 2015.
  2. doi:10.17516/1997-1389-0264
    .
  3. PMID 34139065
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  4. ^ "X-Shining Thermostable Luciferase".
  5. PMID 3072883
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  6. PMID 9806891
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  7. PMID 2871634
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  8. S2CID 23916875
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  9. PMID 8749369
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  10. ^
    PMID 15665092
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  11. PMID 11747464
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  12. PMID 8703001
    .
  13. doi:10.1021/cen-v077n003.p065
    .
  14. ^ a b Knivett V (2009). "Lighting the way". EE Times. Archived from the original on 2012-10-05. Retrieved 2011-09-18.
  15. ^ General Electric TP-110, p. 23, table.
  16. PMID 12117758
    .
  17. ^ a b "Introduction to Bioluminescence Assays". Promega Corporation. Archived from the original on 2010-08-14. Retrieved 2009-03-07.
  18. PMID 2785354
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  19. PMID 3407940
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  20. ^
    PMID 17355205
    .
  21. ^ Meisenheimer PL, O'Brien MA, Cali JJ (September 2008). "Luminogenic enzyme substrates: The basis for a new paradigm in assay design" (PDF). Promega Notes. 100: 22–26. Archived from the original (PDF) on 2009-03-06. Retrieved 2008-10-01.
  22. PMID 16564487
    .
  23. PMID 17540326
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  24. PMID 11816060
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  25. PMID 14612492
    .
  26. PMID 16585152
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  27. S2CID 40630078
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  28. PMID 17254764
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External links

This article incorporates text from the public domain Pfam and InterPro: IPR018804
This article incorporates text from the public domain Pfam and InterPro: IPR007959
This article incorporates text from the public domain Pfam and InterPro: IPR018475
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