Luciferase
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Luciferase is an enzyme best known for its function in the bioluminescence of fireflies (Goodsell, 2006). It is classified as an oxidoreductase. An oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the electron donor, to another, the electron acceptor, often using a cofactor. Luciferase uses a luciferin cofactor to trap oxygen, resulting in an electronically excited state that releases a photon of light upon return to the ground state.
In nature, bioluminescence is used for defense, camouflage, feeding, and mating (Thermo Fisher Scientific Inc., 2015). In addition to fireflies, bioluminescence is also utilized by bacteria, fungi, sea anemones, and dinoflagellates. Interestingly, the luciferase protein of each species varies in size and shape, suggesting that the protein has evolved separately in each to perform the same function (Goodsell, 2006).
Luciferase can also be used in the laboratory to study biological systems. One such use is as a gene reporter. Reporter genes are those used to study the genetic regulation of gene expression; luciferase is inserted into the DNA of cell and then measured over time to determine the activity of the pathway (Thermo Fisher Scientific Inc., 2015).)Luciferase is also being used to study cells and protein in vivo; this is especially useful in following the progression of cancer. One of the most interesting applications of this protein is ongoing research by the Glowing Plant Project. This project aims to genetically engineer a plant that can express the luciferase gene and provide a sustainable light source; currently, the project has completed the prototype stage (Glowing Plant, 2015).
Primary Structure
Luciferase is a single-chain polymer comprised of 548 amino acid residues (RCSB Protein Data Bank, 2006).
Secondary Structure
Luciferase is 31% helical (20 helices or 172 residues) and 23% beta sheets (35 strands or 129 residues) (RCSB Protein Data Bank, 2006).
Supersecondary Structure (Motifs)
In the N-terminal domain, there are two β-sheets arranged in a five-layered αβαβα structure and a β-barrel flanking them. The two β-sheets stack on top of one another and are covered on the end by the β-barrel. The C-terminal domain has an α+β structure, in which two short antiparallel β-strands and a 3-stranded mixed β-sheet are packed with three helices at the sides (RCSB Protein Data Bank, 2006).
Tertiary Structure
Luciferase is a globular protein, in which the non-polar amino acid side chains are on the inside away from water and the polar amino acid side chains are on the outside interacting with water. Some of the amino acids on the inside include methionine, phenylalanine, proline, and alanine. Some of the amino acids likely to be on the outside include serine, tyrosine, threonine, asparagine, and glutamine (RCSB Protein Data Bank, 2006).
Domains
Luciferase has two distinct domains: the N-terminal and the C-terminal. The domains are connected by a 'flexible hinge'. The N-terminal is from residues 4-436 and the C-terminal is from residues 440-548. The C-terminal forms a 'lid' over the β-barrel of the N-terminal (RCSB Protein Data Bank, 2006).
Luciferase binds to luciferin in order to emit its bioluminescence. Specifically, to glow, the luciferin substrate must be in its oxygenated form complexed with AMP called DLSA (Nakatsu et al., 2006). The binding of DLSA is contained in a hydrophobic pocket of α8, β12, β13, β14, β15 and a loop. It is held in place by Van der Waals forces and hydrogen bonding to water.
Active SiteAs an oxioreductase, luciferase catalyzes the transfer of electrons from an electron donor to an electron acceptor. Luciferin, the e- donor, utilizes an ATP cofactor and oxygen to be put into an electronically excited state. Upon oxidation of luciferin into oxyluciferin and AMP (also a complex known as DSLA), a photon of light is emitted as a yellow-green glow as it returns to its ground state (Goodsell, 2006).
The color of light emitted by the binding of luciferase and luciferin is dependent upon the amino acids holding the ligand in the enzyme's active site (Nakatsu et al., 2006). The two most influential residues in the wavelength of light emitted are 286 and 288.
In fireflies, serine 286 causes a greenish-yellow light to be emitted. This is due to isoleucine 288 moving closer to the DSLA, creating hydrogen bonds between Ser286, Tyr257, and Asn231 via water. In other insects, however, Ser286 can be replaced by asparagine 286, resulting in a reddish glow. This is due to weaker hydrogen bonds linking the residues of the active site, resulting in lower energy and thus a different wavelength of light.
Glowing Plant. (2015). Glowing plant. Retrieved November 12, 2015, from http://www.glowingplant.com/
Goodsell, D. (2006). Luciferase. In RCSB Protein Data Bank. http://dx.doi.org/10.2210/rcsb_pdb/mom_2006_6
Nakatsu, T., Ichiyama, S., Hiratake, J., Saldanha, A.,Kobashi, N., Sakata, K., & Kato, H. (2006). Structural basis for the spectral difference in luciferase bioluminescence. Nature,440, 372-376. http://dx.doi.org/10.1038/nature04542
RCSB Protein Data Bank. (2006). Crystal structure of the thermostable Japanese Firefly Luciferase complexed with high-energy intermediate analogue. In RCSB protein data bank. http://dx.doi.org/10.2210/pdb2d1s/pdb
Thermo Fisher Scientific Inc. (2015). Luciferase reporters. Retrieved November 12, 2015, from https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/luciferasereporters.html