Люциферазы: ферменты биолюминесценции и их применение.

Enzyme family

Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. Both words are derived from the Latin word lucifer, meaning "lightbearer", which in turn is derived from the Latin words for "light" (lux) and "to bring or carry" (ferre). Luciferases are widely used in biotechnology, for bioluminescence imaging microscopy and as reporter genes, for many of the same applications as fluorescent proteins. However, unlike fluorescent proteins, luciferases do not require an external light source, but do require addition of luciferin, the consumable substrate.

A variety of organisms regulate their light production using different luciferases in a variety of light emitting reactions. The majority of studied luciferases have been found in animals, including fireflies, and many marine animals such as copepods, jellyfish, and the sea pansy. However, luciferases have been studied in luminous fungi, like the Jack O Lantern mushroom, as well as examples in other kingdoms including bioluminescent bacteria, and dinoflagellates.

The luciferases of fireflies – of which there are over 2000 species – and of the other Elateroidea (click beetles and relatives in general) are diverse enough to be useful in molecular phylogeny. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea. One well studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8.

Newer luciferases have recently been identified that, unlike other luciferases, are naturally secreted molecules. One such example is the Metridia coelenterazine dependent luciferase (MetLuc, ) that is derived from the marine copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein containing an N terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.

Bacterial bioluminescence is seen in Photobacterium species, Vibrio fischeri, Vibrio haweyi, and Vibrio harveyi. Light emission in some bioluminescent bacteria utilizes 'antenna' such as lumazine protein to accept the energy from the primary excited state on the luciferase, resulting in an excited lulnazine chromophore which emits light that is of a shorter wavelength (more blue), while in others use a yellow fluorescent protein (YFP) with flavin mononucleotide (FMN) as the chromophore and emits light that is red shifted relative to that from luciferase.

Dinoflagellate luciferase is a multi domain eukaryote protein, consisting of an N terminal domain, and three catalytic domains, each of which preceded by a helical bundle domain. The structure of the dinoflagellate luciferase catalytic domain has been solved. The core part of the domain is a 10 stranded beta barrel that is structurally similar to lipocalins and FABP. The helical bundle domain has a three helix bundle structure that holds four important histidines that are thought to play a role in the pH regulation of the enzyme. Nearly all of the energy input into the reaction is transformed into light. The reaction is 80% to 90% efficient. In comparison, the incandescent light bulb only converts about 10% of its energy into light and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light. In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or an optical microscope with a CCD camera. This allows observation of biological processes. Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and therefore the bioluminescent signal is virtually background free. Therefore, as little as 0.02 pg can still be accurately measured using a standard scintillation counter. In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. Additionally, proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two step luciferase assays. Such substrates have been used to detect caspase activity and cytochrome P450 activity, among others. Luciferase can act as an ATP sensor protein through biotinylation. Biotinylation will immobilize luciferase on the cell surface by binding to a streptavidin biotin complex. This allows luciferase to detect the efflux of ATP from the cell and will effectively display the real time release of ATP through bioluminescence. Luciferase can additionally be made more sensitive for ATP detection by increasing the luminescence intensity by changing certain amino acid residues in the sequence of the protein. Whole organism imaging (referred to as in vivo when intact or, otherwise called ex vivo imaging for example of living but explanted tissue) is a powerful technique for studying cell populations in live plants or animals, such as mice. Different types of cells (e. g. bone marrow stem cells, T cells) can be engineered to express a luciferase allowing their non invasive visualization inside a live animal using a sensitive charge couple device camera (CCD camera). This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models. However, environmental factors and therapeutic interferences may cause some discrepancies between tumor burden and bioluminescence intensity in relation to changes in proliferative activity. The intensity of the signal measured by in vivo imaging may depend on various factors, such as D luciferin absorption through the peritoneum, blood flow, cell membrane permeability, availability of co factors, intracellular pH and transparency of overlying tissue, in addition to the amount of luciferase. Luciferase is a heat sensitive protein that is used in studies on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for using luciferase continue to expand.