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Luciferase 4M46Luciferase 4M46
Function4M46's main function is to control a light-emitting reaction that occurs in fireflies. It is naturally present in Lampyris Turkestanicus and emits a green light. Luciferase 4M46's catalytic action involves helping D-Luciferin binding ATP, which is the first step of several reactions that lead an excited oxyluciferin to release light. This mechanism is essential for the reproduction of fireflies, but is also often used in genetic engineering as reporter gene. This enzyme is specific of two reactions : Luciferase can operate in a bioluminescence pathway (which is the most known) or in a CoA-ligase pathway. In both case, luciferase initially acts as a catalyst for the reaction of adenylation with MgATP. In the bioluminescence pathway, the oxidation of luciferin to oxyluciferin in the presence of oxygen, ATP and Mg2+ leads to the emission of a photon and thus to a yellow-green light. [3] [4] But, in the CoA-ligase pathway, CoA can substitute AMP to form luciferyl CoA. Moreover, fatty acyl-CoA synthetase and ATP activate fatty acids, then the displacement of AMP with CoA takes place. Luciferase can substitute fatty acyl-CoA synthetase and convert long-chain fatty acids into fatty-acyl CoA for beta oxidation thanks to their comparable activities. It occurs when the chirality of luciferin is L(+)-Luciferin. [3] [4] RelevanceLuciferases are used a lot as reporter genes in the study of gene promoter sequences. They are at the origin of the ATPmetric method. [1] They are used as reporter gene modify. In molecular cloning, the luciferase gene can be coupled with a gene of interest to guarantee the effectiveness of its insertion since the reporter gene, the luciferase gene, and the gene of interest are fused. They will always be expressed together. The luminescence observed is therefore directly related to gene expression. [1] So, the firefly luciferase gene is frequently used as a reporter of genetic function, or for producing recombinant luciferase. For example, mutant plasmids were transformed to competent cells of Escherichia coli BL21 for expression of protein and purification using affinity chromatography. For this, cDNA encoding for L. turkestanicus luciferase was isolated by reverse transcription-polymerase chain reaction, cloned, and functionally expressed in Escherichia coli. [2] Another example of use is that the luciferase-catalyzed bioluminescence reaction is used to determine aquantity of ATP, for example to analyze cell proliferation or cytotoxicity of specific cells. The light emitted during this luciferase-catalyzed reaction is due to the release of a photon when one of the reaction intermediates passes from an excited to a relaxed state. In addition to the substrate, D-luciferin, three cofactors must be present to allow the reaction : ATP, oxygen and Mg2+ are required for light emission. The concentration of the reagents thus influences the activity of the luciferase. The role of ATP as a cofactor in the bioluminescence reaction is thus used for its determination since when the concentration of ATP is limiting, the intensity of light is proportional to the concentration of this molecule. [1] [3] Structural highlightsLuciferase 4M46 is constituted that are folded around each other. The protein is a monomer made of one unique chain of 581 amino-acid residues that builds alternatively the two domains. Domain 1 (N-terminal), which is the largest includes residues 1-86, 206-401 and 457-581 while domain 2 (C-terminal) contains all the others. The C-terminal domain is much more condensed than its counterpart. When the catalysis transcurs, the two domain close the protein thanks to a flexible hinge (residues 435-441) between the two, so that ATP, bound in the hydrophobic pocket, is not hydrolysed. The amino-acids sequences surrounding the separation of the two domains undergo very few modifications from one luciferase to another. No mechanical observation have confirmed this theory, but it is possible that the active site is located between the two domains. [5]
The color of light emitted is affected in all types of luciferase by a loop formed by the . This loop contains notable hydrogen bonds that are responsible for its activity. According to the different amino acids present in the loop, the color changes from one luciferase to another (yellow to green colors usually). The flexibility of this loop is very important for the emission of light. Studies have shown that mutating it with amino acids that change the conformation leads to incapacity of the enzyme.
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ReferencesReferences
[1] Satoshi Inouye, « Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions », Cell. Mol. Life Sci., no 67, 27 octobre 2010, p. 387-404
[2] Mojtaba Mortazavi1, Saman Hosseinkhani1*, Khosro Khajeh1, Bijan Ranjbar2, and A. Rahman Em, «Spectroscopic and functional characterization of Lampyris turkestanicus luciferase: a comparative study », Acta Biochim Biophys Sin 2008, 40: 365-374
[3] Yuichi Oba, « Firefly luciferase is a bifonctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase », FEBS, no 540, 21 mars 2003, p. 251-254
[4] Oba Y, Ojika M, Inouye S (Apr 2003). "Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase". FEBS Letters. 540 (1–3): 251–54
[5] Conti E, Franks NP, Brick P (Mar 1996). "Crystal structure of firefly luciferase throws light on a superfamily of adenylate forming enzymes". Structure. 4 (3): 287-98