Sandbox Reserved 1731

From Proteopedia

Photinus pyralis luciferase

Luciferases are enzymes that catalyzes the light-producing chemical reactions of bioluminescent organisms, such as fireflies and bacteria. Photinus pyralis luciferase, which is the specific luciferase protein found in the common eastern firefly, causes an enzymatic reaction between the enzyme (luciferase) and the substrate (luciferin). This reaction is an oxidation process with molecular oxygen and its conversion into chemical energy, which causes the bursts of light ¹.

Structure

The overall structure of luciferase is an asymmetrical monomer. It is composed of two domains specified as the N-terminal and C-terminal. The consists of a beta-barrel and two beta-sheets flanked by alpha-helices to form a five layer structure α-β-α-β-α structure. The consists of five beta-strands and three alpha-helices, which is folded into a compact structure that is connected to the N-terminus domain by a disordered loop. A beta barrel is one singular beta sheet, where the beta strands run antiparallel to one another. Luciferase is composed of 550 amino acids residues in a single polypeptide chain with a peroxisome targeting signal sequence of -Ser-Lys-Leu (-SKL) at C-terminus. The overall structure of luciferase contains ².

Function

Luciferase proteins act as enzymes to to produce bursts of light through oxidation reactions of the substrate, luciferin. The reaction formula is:

ATP + firefly D-luciferin + O₂ ⇌ AMP + CO₂ + diphosphate + firefly oxyluciferin + hnu ³

The reaction begins with the formation of an acid anhydride between the carboxylic group of D-firefly luciferin and AMP, with the release of diphosphate. An oxygenation follows, with the release of the AMP group group and formation of a very short-lived peroxide that cyclizes into a dioxetanone structure, which collapses, releasing a CO₂ molecule. The spontaneous breakdown of the dioxetanone (rather than the hydrolysis of the hydrolysis of the adenylate) releases the energy (about 50 kcal/mole) that is necessary to generate the excited state of oxyluciferin. The excited luciferin then emits a photon or light (hnu), returning to its ground state. The enzyme has a secondary acyl-CoA ligase activity when acting on L-firefly luciferin ^{4 5}.

Biology

Bioluminescence provides a variety of benefits to the many different organisms who utilize it, such as multiple kinds of bacteria and species of firefly. The eastern common firefly (Photonis pyralis) utilize the bioluminescent characteristic in mating. They are nocturnal as a species use a discrete pulse of bioluminescence to locate their mates during the night. Both sexes of the species produce flash signals that are timed precisely to relay information about the insects species identity and sex. The brightness of the bioluminescent pulse is dependent on the amount of ATP in the cells. If the insect is not producing enough ATP in the cells, the pulses of light will be duller ⁶.

Bioluminescence is also used in a laboratory setting. The most common laboratory use for luciferase is as a detector for a variety of tests. One example of this is the detection of bacteriuria, where if samples were "luciferase-positive", they contained high levels of ATP, proving the presence of the bacteria ⁷. Luciferase is also be used in other laboratory tests such as, quantitative analysis of viral infections as well as observing the effectiveness of new antimicrobial therapies ⁸. Equally important, luciferase is also used for in vivo cell imaging where the cell of interest is genetically modified to produce the enzyme ⁹. Lastly, luciferase is one of the more commonly used reporter enzymes used to study eukaryotic gene expression ¹⁰. The usage of luciferase in the lab is an easy organic option for an indicator or detector for a variety of tests.

References

1. de Wet, J. R.; Wood, K. V.; Helinski, D. R.; DeLuca, M. Cloning of Firefly Luciferase Cdna and the Expression of Active Luciferase in Escherichia Coli. Proc. Natl. Acad. Sci. U.S.A. 1985, 82 (23), 7870–7873.

2. Baldwin, T. O. Firefly Luciferase: The Structure Is Known, but the Mystery Remains. J. Am. Chem. Soc. 1996, 4 (3), 223–228.

3. de Wet, J. R.; Wood, K. V.; DeLuca, M.; Helinski, D. R.; Subramani, S. Firefly Luciferase Gene: Structure and Expression in Mammalian Cells. Mol. Cell. Biol. 1987, 7 (2), 725–737.

4. Inouye, S. Firefly Luciferase: An Adenylate-Forming Enzyme for Multicatalytic Functions. Cell. Mol. Life. Sci. 2009, 67 (3), 387–404.

5. Marques, S. M.; Esteves da Silva, J. C. Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions. IUBMB. Life. 2009, 61 (1), 6–17.

6. Lewis, S. M.; Cratsley, C. K. Flash Signal Evolution, Mate Choice, and Predation in Fireflies. Annu. Rev. Entomol. 2008, 53 (1), 293–321.

7. Thore, A.; Anséhn, S.; Lundin, A.; Bergman, S. Detection of Bacteriuria by Luciferase Assay of Adenosine Triphosphate. JCM. 1975, 1 (1), 1–8.

8. Luker, K. E.; Luker, G. D. Applications of Bioluminescence Imaging to Antiviral Research and Therapy: Multiple Luciferase Enzymes and Quantitation. Antiviral Res. 2008, 78 (3), 179–187.

9. J. Syed, A.; C. Anderson, J. Applications of Bioluminescence in Biotechnology and Beyond. Chem. Soc. Rev. 2021.

10. Thompson, J. F.; Hayes, L. S.; Lloyd, D. B. Modulation of Firefly Luciferase Stability and Impact on Studies of Gene Regulation. Gene. 1991, 103 (2), 171–177.