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 links

Examples[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 482 nm). 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 510 nm). The catalyzed reaction is:[4]

coelenterazine + O2 coelenteramide + CO2 + photon of light

Bacterial[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 Nterminal 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 + light

Light 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 + light

In 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 dari bahasa Belanda: genetica, adaptasi dari bahasa Inggris:genetics,
dibentuk dari kata bahasa Yunani ,genno, yang berarti "melahirkan") adalah
cabang biologi yang mempelajari pewarisan sifat padaorganisme maupun suborganisme
(seperti virus dan prion). Secara singkat dapat juga dikatakan bahwa genetika adalah ilmu
tentang gen dan segala aspeknya.

Mechanism for luciferase.[2]

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

Lucifer

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 Nterminal 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 = 550 nm) 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: Lluciferin 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 cofactors, 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 Indah

Tuesday, 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 aequorinyang aslinya berasal
dari ubur-ubur laut, yang dikenal dengan GreenFluorosceint
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.
Genpewartatransformasigendanlokalisasiprotein
Tujuan dari pembuatan organismetransgenikyang 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
geneatau 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.colimisalnya, maka sel E.
coliyang 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 trackingatau 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.
Takhanyaberpendarhijau
Mengapa 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 chromophoreyang berperan sangat penting dalam
proses perpendaran hijau. Chromophoreini 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 chromophoredengan
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 trackingtidak 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 glowin-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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