Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. The name is derived from Lucifer, the root of which means 'light-bearer' (lucem ferre). One example is the firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis.[1] "Firefly luciferase" as a laboratory reagent often refers to P. pyralis luciferase although recombinant luciferases from several other species of fireflies are also commercially available.Contents [hide] 1 Examples 1.1 Firefly and click beetle 1.2 Sea pansy 1.3 Bacterial 1.4 Dinoflagellate 1.5 Copepod 2 Mechanism of reaction 3 Bifunctionality 4 Structure 5 Spectral differences in bioluminescence 6 Regulation 7 Applications 8 See also 9 References 10 External linksExamples[edit source| editbeta]A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the fireflies,[2] although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures.Firefly and click beetle[edit source| editbeta]The luciferases of fireflies - of which there are over 2000 species - and of the Elateroidea (fireflies, 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.[3]Sea pansy[edit source| editbeta]Also well studied is the luciferase from the sea pansy, Renilla reniformis. In this organism, the luciferase (Renilla-luciferin 2-monooxygenase) is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510nm). The catalyzed reaction is:[4] coelenterazine + O2 coelenteramide + CO2 + photon of lightBacterial[edit source| editbeta]Bacterial bioluminescence is seen in Photobacterium species, Vibrio fischeri, haweyi, and harveyi. Light emission in some utilize '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 FMN as the chromophore and emits light that is red-shifted relative to that from luciferase.[5]Dinoflagellate[edit source| editbeta]Dinoflagellate luciferase is a multi-domain 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.[6] The core part of the domain is a 10 stranded beta barrel that is structurally similar to lipocalins and FABP.[6] The N-terminal domain is conserved between dinoflagellate luciferase and luciferin binding proteins (LBPs). It has been suggested that this region may mediate an interaction between LBP and luciferase or their association with the vacuolar membrane.[7] 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.[6]Copepod[edit source| editbeta]Newer luciferases have recently been identified that, unlike other luciferases above, are naturally secreted molecules. One such example is the Metridia 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.[8]Mechanism of reaction[edit source| editbeta]The chemical reaction catalyzed by firefly luciferase takes place in two steps: luciferin + ATP luciferyl adenylate + PPi luciferyl adenylate + O2 oxyluciferin + AMP + lightLight is emitted because the reaction forms oxyluciferin in an electronically excited state. The reaction releases a photon of light as oxyluciferin returns to the ground state.Luciferyl adenylate can additionally participate in a side reaction with O2 to form hydrogen peroxide and dehydroluciferyl-AMP. About 20% of the luciferyl adenylate intermediate is oxidized in this pathway.[9]The reaction catalyzed by bacterial luciferase is also an oxidative process: FMNH2 + O2 + RCHO FMN + RCOOH + H2O + lightIn the reaction, a reduced flavin mononucleotide oxidizes a long-chain aliphatic aldehyde to an aliphatic carboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to the product FMN to emit blue-green light.[10]Nearly all of the energy input into the reaction is transformed into light. The reaction is 80%[11] to 90%[12] efficient. As a comparison, the incandescent light bulb only converts about 10% of its energy into light.[13] and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.[12]Firefly luciferase generates light from luciferin in a multistep process. First, D-luciferin is adenylated by MgATP to form luciferyl adenylate and pyrophosphate. After activation by ATP, luciferyl adenylate is oxidized by molecular oxygen to form a dioxetanone ring. A decarboxylation reaction forms an excited state of oxyluciferin, which tautomerizes between the keto-enol form. The reaction finally emits light as oxyluciferin returns to the ground state.[2]

Genetika(dipinjam daribahasa Belanda:genetica, adaptasi daribahasa Inggris:genetics, dibentuk dari katabahasa Yunani,genno, yang berarti "melahirkan") adalah cabangbiologiyang mempelajari pewarisan sifat padaorganismemaupun suborganisme (sepertivirusdanprion). Secara singkat dapat juga dikatakan bahwa genetika adalah ilmu tentanggendan segala aspeknya.

Mechanism for luciferase.[2]

Luciferase has two modes of enzyme activity: bioluminescence activity and CoA synthetase activity.[14]

Bifunctionality[edit source| editbeta]Luciferase can function in two different pathways: a bioluminescence pathway and a CoA-ligase pathway.[15] In both pathways, luciferase initially catalyzes an adenylation reaction with MgATP. However, in the CoA-ligase pathway, CoA can displace AMP to form luciferyl CoA.Fatty acyl-CoA synthetase similarly activates fatty acids with ATP, followed by displacement of AMP with CoA. Because of their similar activities, luciferase is able to replace fatty acyl-CoA synthetase and convert long-chain fatty acids into fatty-acyl CoA for beta oxidation.[15]Structure[edit source| editbeta]The protein structure of firefly luciferase consists of two compact domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is composed of two -sheets in an structure and a barrel. The two -sheets stack on top of each other, with the -barrel covering the end of the sheets.[2]The C-terminal domain is connected to the N-terminal domain by a flexible hinge, which can separate the two domains. The amino acid sequences on the surface of the two domains facing each other are conserved in bacterial and firefly luciferase, thereby strongly suggesting that the active site is located in the cleft between the domains.[16]During a reaction, luciferase has a conformational change and goes into a closed form with the two domains coming together to enclose the substrate. This ensures that water is excluded from the reaction and does not hydrolyze ATP or the electronically excited product.[16]

Diagram of the secondary structure of firefly luciferase. Arrows represent -strands and circles represent -helices. The locations of each of the subdomains in the sequence of luciferase is shown in the bottom diagram.[16]Spectral differences in bioluminescence[edit source| editbeta]Firefly luciferase bioluminescence color can vary between yellow-green (max = 550nm) to red (max = 620).[17] There are currently several different mechanisms describing how the structure of luciferase affects the emission spectrum of the photon and effectively the color of light emitted.One mechanism proposes that the color of the emitted light depends on whether the product is in the keto or enol form. The mechanism suggests that red light is emitted from the keto form of oxyluciferin, while green light is emitted from the enol form of oxyluciferin.[18][19] However, 5,5-dimethyloxyluciferin emits green light even though it is constricted to the keto form because it cannot tautomerize.[20]Another mechanism proposes that twisting the angle between benzothiazole and thiazole rings in oxyluciferin determines the color of bioluminescence. This explanation proposes that a planar form with an angle of 0 between the two rings corresponds to a higher energy state and emits a higher-energy green light, whereas an angle of 90 puts the structure in a lower energy state and emits a lower-energy red light.[21]The most recent explanation for the bioluminescence color examines the microenvironment of the excited oxyluciferin. Studies suggest that the interactions between the excited state product and nearby residues can force the oxyluciferin into an even higher energy form, which results in the emission of green light. For example, Arg 218 has electrostatic interactions with other nearby residues, restricting oxyluciferin from tautomerizing to the enol form.[22] Similarly, other results have indicated that the microenvironment of luciferase can force oxyluciferin into a more rigid, high-energy structure, forcing it to emit a high-energy green light.[23]Regulation[edit source| editbeta]D-luciferin is the substrate for firefly luciferases bioluminescence reaction, while L-luciferin is the substrate for luciferyl-CoA synthetase activity. Both reactions are inhibited by the substrates enantiomer: L-luciferin and D-luciferin inhibit the bioluminescence pathway and the CoA-ligase pathway, respectively.[14] This shows that luciferase can differentiate between the isomers of the luciferin structure.L-luciferin is able to emit a weak light even though it is a competitive inhibitor of D-luciferin and the bioluminescence pathway.[24] Light is emitted because the CoA synthesis pathway can be converted to the bioluminescence reaction by hydrolyzing the final product via an esterase back to D-luciferin.[25]Luciferase activity is additionally inhibited by oxyluciferin [26] and allosterically activated by ATP. When ATP binds to the enzymes two allosteric sites, luciferases affinity to bind ATP in its active site increases.[17]Applications[edit source| editbeta]Luciferase can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. Mice, silkworms, and potatoes are just a few of the organisms that have already been engineered to produce the protein.[27]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 modified optical microscopes. This allows observation of biological processes.[28] Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and therefore virtually background-free fluorescence. [29] Therefore, as little as 0.02pg can still be accurately measured using a standard scintillation counter. [30]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.[31] 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.[28][31]Luciferase can also be used to detect the level of cellular ATP in cell viability assays or for kinase activity assays.[31][32] 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.[33] Luciferase can additionally be made more sensitive for ATP detection by increasing the luminescence intensity through genetic modification.[34]Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice.[35] 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.[36][37] 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.[38]Luciferase can be used in blood banks to determine if red blood cells are starting to break down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. 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

GFP, Gen Pewarta yang Berpendar IndahTuesday, 23 October 2007(http://www.biotekindonesia.com)Di dalam majalah ilmiah ataupun berita di televisi, mungkin anda pernah melihat tikus percobaan berpendar hijau, atau daun tembakau bercahaya hijau di kegelapan. Pemandangan yang tidak biasa dan membuat terpesona ini tidak akan kita jumpai pada tikus ataupun tembakau alami. Para ilmuwan memang sengaja membuatnya. Namun, bukan untuk tujuan keindahan visual semata.Pendar cahaya hijau yang merupakan cahaya floresens ini dihasilkan oleh sebuah protein aequorin yang aslinya berasal dari ubur-ubur laut, yang dikenal dengan Green Fluorosceint Protein (GFP). Gen penyandi protein ini dicangkokkan pada genom tikus ataupun sel tembakau dengan proses rekayasa genetika, sehingga makhluk hidup yang telah dimasukkan gen ini akan berpendar hijau.Gen pewarta transformasi gen dan lokalisasi proteinTujuan dari pembuatan organisme transgenik yang dapat berpendar pada dasarnya adalah untuk pemantauan suatu proses eksperimen atau biokimia di dalam sel tubuh makhluk hidup.Untuk proses eksperimen rekayasa genetika misalnya, konfirmasi transformasi dan ekspresi suatu gen asing ke dalam sel inang sangatlah penting. Transformasi adalah proses memasukkan atau mencangkokkan gen asing ke dalam sel makhuk hidup lain, dapat berupa sel bakteri, ragi, tumbuhan, ataupun mamalia.Bagi para peneliti, sangatlah penting untuk dapat mengetahui dengan cepat, apakah proses transformasi itu telah berlangsung baik atau tidak, dan apakah gen target dapat diekspresikan tanpa masalah. Dari itu, di dalam molekuler biologi dikenal reporter gene atau gen pewarta. Yaitu gen yang dapat memberitahukan dengan jelas pada para peneliti bahwa proses transformasi telah berjalan dengan sukses. Gen yang menghasilkan protein yang dapat berpendar hijau atau GFP inilah yang banyak dipakai oleh para ilmuwan sebagai gen pewarta.Gen asing target yang ingin dicangkokkan ke dalam suatu sel organisme biasanya digabungkan dengan gen GFP dalam bentuk gen kimera atau gen gabungan, sehingga nanti akan dihasilkan protein baru fungsional (yang menjadi sifat baru organisme tersebut) dalam bentuk protein gabungan dengan GFP. Jadi, jika gen yang digabung dengan gen pewarta berhasil masuk dan fungsional di dalam sel bakteri E. coli misalnya, maka sel E. coli yang berpendar hijau di kegelapan adalah bakteri transgenik yang fungsional yang membawa sifat baru. Proses penapisan klon transgenik yang positif menjadi lebih cepat dan mudah.Dibandingkan dengan gen pewarta lain yang kebanyakan enzim yang memerlukan substrat untuk menghasilkan warna atau pendar cahaya, GFP adalah gen pewarta yang menarik sekaligus mudah dalam hal visualisasi, karena untuk berpendar GFP sama sekali tak memerlukan substrat. GFP menghasilkan sinar hijau fluoresens secara instrinsik, ketika diberi sinar eksitasi pada panjang gelombang biru sekitar 395 nanometer. Jadi hanya dibutuhkan lampu UV gelombang panjang atau sinar biru untuk dapat mendeteksinya dalam kegelapan.GFP juga menjadi gen pewarta idola dalam hal pencitraan proses tracking atau lokalisasi suatu protein. Karena tidak perlu penambahan substrat dan mudah divisualisasi, GFP dapat diaplikasikan untuk memantau jejak protein, kapan dan dimana suatu gen terinduksi menjadi suatu protein, dalam kondisi sel masih hidup.Pada tumbuhan tembakau yang berpendar hijau misalnya. Gen GFP diekspresikan pada virus mosaik yang biasa menyerang tumbuhan tembakau. Banyak hal yang masih belum diketahui tentang interaksi virus ini dengan tembakau. Dalam proses terinfeksinya tembakau dengan virus ini sampai tembakau menjadi sakit lalu mati, para peneliti tanaman tidak mengetahui lokasi awal timbulnya virus dan penyebarannya. Dengan memasukkan virus mosaik yang mengekspresikan GFP, maka tempat penyebaran virus dapat terpantau hanya dengan membawa tanaman tembakau ini ke ruang gelap dan menyinarinya dengan lampu UV secara periodik.Tak hanya berpendar hijauMengapa GFP dapat berpendar hijau? GFP adalah protein yang merupakan polimer dari 238 asam amino dengan berat molekul sekitar 27 Kilo Dalton. Di dalam protein ini ada gugus yang disebut chromophore yang berperan sangat penting dalam proses perpendaran hijau. Chromophore ini adalah kelompok tiga residu asam amino di posisi 65 (Serin), 66 (Tirosin), dan 67 (Glisin). Ketika dikenai energi cahaya biru atau UV maka pada gugus ini akan terjadi reaksi oksidasi. Energi yang diserap membuat elektron- elektron di dalam gugus ini tereksitasi dan menghasilkan energi yang lebih rendah yaitu energi cahaya hijau.Pengetahuan para ilmuwan yang lengkap tentang molekuler dan struktur dari GFP ini membuat para ilmuwan dapat merekayasa protein ini menjadi beberapa mutan. Sekarang tidak hanya protein yang dapat berpendar hijau yang digunakan sebagai gen pewarta. Tetapi juga protein berpendar biru, merah, atau kuning berhasil ditemukan oleh para ilmuwan sebagai turunan dari GFP.Ilmuwan mengganti asam amino di gugus chromophore dengan asam amino lain dengan proses mutasi gen. Misalnya asam amino ke 66 (Tirosin) disubstitusi dengan asam amino Histidin, mutasi ini menyebabkan protein menghasilkan warna pendar biru bukan hijau. Subtitusi asam amino ke 203 (Treonin), yang posisinya dalam kristal GFP dekat chromophore, dengan Tirosin menghasilkan protein yang berpendar kuning. Gen pewarta turunan GFP yang menghasilkan berbagai pendar warna ini memudahkan para peneliti untuk melakukan pemantauan beberapa proses biokimia secara bersamaan, sehingga informasi yang diperoleh dapat lebih cepat dan lebih lengkap.Karena sinar UV relatif berbahaya untuk sel makhluk hidup, maka para ilmuwan juga merekayasa protein ini (juga dengan proses mutasi gen), sehinga hanya dapat dieksitasi oleh energi cahaya gelombang panjang. Dengan demikian proses deteksi transformasi ataupun tracking tidak berbahaya bagi sel makhluk hidup, karena tidak perlu terpapar dengan sinar UV.Pencitraan dengan menggunakan GFP sebagai gen pewarta memang bukan hanya untuk keindahan visual semata. Yang lebih penting dari itu adalah banyak proses dan pengetahuan baru biologi yang diperoleh atas jasanya sebagai gen pewarta.

An image of a tobacco plant which has been genetically engineered to express a gene taken from fireflies (specifically: Photinus pyralis) which produces luciferase. The image is an "autoluminograph" produced by placing the plant directly on a piece of Kodak Ektachrome 200 film. When the plant is watered with a luciferin containing nutrient medium, tissue specific luminescence is observed. It is the first representation of a transgenic multicellular organism expressing bioluminescence. This image was first published in a November 1986 issue of the journal Science in a paper titled "Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants". [1] by David W. Ow, Keith V. Wood, Marlene DeLuca, Jeffrey R. de Wet, Donald R. Helinski and Stephen H. Howell. The research was funded by grants from the US Dept. of Agriculture and the National Science Foundation.Image taken by Keith Wood (of DeLuca lab) for Science Magazine. Permission to use on Wikipedia has been granted by Science Magazine

Bioluminescence is the process that makes these creatures produce naturally-occurring light from their bodies.The team start off by getting glowing protein enzymes called Luciferase, from the genes of fireflies or from bacteria. They then use software called Genome Compiler to make it possible for the plants to read what those genes are.The genes are then made in labs and shipped to the team in California.

This chart from the Glowing Plant project shows how the team creates the glow-in-the-dark plants, which could be used to replace electrical street lighting

The Californian scientists have tested their technology on a range of plants in their DIY biolab. Anyone who pledges money to the project's Kickstarter campaign can get a glow-in-the-dark rose, or be given the chance to buy one before anyone else

Evans and his team put these genes into liquid agrobacteria and the bacteria is poured over the plants.Agrobacteria is able to transfer genes into plants, and when these glowing genes are added, they are transferred to the plants, which makes them glow-in-the-dark.To create these genes, the scientists have had to redesign the DNA sequence.They have successfully managed to create small glowing plants and are now asking for extra funding, via a Kickstarter campaign, to use the technology on larger plants and trees.The campaign ends on 7 June.So far it has had more than 5,000 backers and raised over 183,000

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