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Function

4M46'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 a excited oxyluciferin to release light. This mechanism is essential for the reproduction of fireflies, but is also often use in genetic engineering as reporter gene (cf Relevance).

Relevance

Luciferases are widely used as reporter genes in the study of gene promoter sequences and are at the origin of the ATPmetric method. They are use 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. So the firefly luciferase gene is frequently used as a reporter of genetic function, or for producing of recombinant luciferase. For example, mutant plasmids were transformed to competent cells of Escherichia coli BL21 for the expression of protein and the 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. Other example of use is that the luciferase-catalyzed bioluminescence reaction is used to determine the quantity 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, so 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.

Structural highlights

Luciferase 4M46 is constituted that are folded around each other. The protein is a monomer made of one unique chain that builds alternatively the two domains. Domain 1 includes residues 1-86, 206-401 and 457-581 while domain 2 contains all the others.