Bioluminescence, the emission of light from a biological process without releasing heat, is found in various living organisms including bacteria, fireflies, beetles, fungus and different marine organisms. Luciferase is an enzyme that catalyzes a bioluminescent reaction by the oxidation of luciferin, in the presence of Mg2+ and ATP, to oxyluciferin and releases energy in the form of light. Luciferase assays are used in biological and clinical applications, where it is the main analytical platform for in vivo imaging, cell proliferation, and protein folding and secretion. The luciferase enzyme consists of two domains: a large N-terminal domain (4-436 residues) that is connected to a small C-terminal domain (440-544) by a flexible loop that function as a hinge. In the substrate-free enzyme, the two domains are separated by a large cleft that houses the active site that closes once the substrates are bound. Even though all luciferases catalyze the same chemical reaction, they emit light of different colors usually in the green-yellow region. Several hypothesis relate the color difference to amino acid residues surrounding the active site, nevertheless the color emission mechanism remains unresolved.
