Proteopedia - User contributions [en]

Sandbox Reserved 951

2015-01-08T23:02:21Z

Amandine Schmit:

<Structure load='1LCI' size='350' frame='true' align='right' caption='Crystal structure of firefly luciferase' scene='Insert optional scene name here' />

==Biological context==
Firefly, also named Photinus pyralis, is a bioluminescent insect. They are able to produce light by a very energetic process, in order to attract its mate. The enzyme responsible of this light producing is luciferase, also known as luciferin-4-monooxygenase (EC: 1.13.12.7). It has various applications in the biotechnology field. In fact, it is used in chemical biology and drug assays. For example, they help to detect protein-protein interactions, to track cells in vivo in order to analysis in real-time the development of a disease in molecular level, to monitor the transcriptional and post-transcriptional regulation of specifics gens, to control apoptosis, to label cancer cells, to detect environmental contamination, to follow protein trafficking, … As there is no light production by mammals, luciferase is a really a great tool for researchers. But this light emission depends on the environmental conditions. Luciferase acts also as a ligase.
Thus, this oxydo-reductase is involved in severals reactions.
=====Light emission=====
Of course, the most known reaction of luciferase is the light emission. In this reaction, luciferase firstly synthetized luciferin-AMP from luciferin and ATP, using a Mg2+ ion to offset the negative charges of the phosphate groups. Then, luciferase turns the luciferin-AMP into oxyluciferin in an excited state thanks to a dioxygen. This step releases AMP and CO2. The excited oxyluciferin relaxes and looses a photon so light is emitted.
The wavelength of the light can vary with the pH : at the physiological pH, the emitted light is green and at a lower pH, the color is red.
=====Fatty-acyl-CoA synthesis=====
This reaction uses similar reaction but instead of taking luciferin as ligand, it takes fatty acids. Using ATP-Mg2+, it firstly forms fatty-acyl-AMP. And then, CoA-SH attacks the carboxylic group of fatty-acyl-AMP and so forms fatty-acyl-CoA.
=====Luciferin & Coenzyme A=====
It has been found that the reaction between luciferin and CoA is possible, forming in a first step luciferin-AMP and then luciferin-CoA. This reaction leads to an interesting biological phenomenon : when this reaction occurs in parallel of light emission reaction, we don't have a flash of light but a continuous light emission. This is because luciferin-AMP is a competitive inhibitor of the reaction whereas the luciferin-CoA is not. So the inhibition is deleted and the reaction continue to occur.<ref>PMID:18949818</ref>

== Global Structure ==
Luciferase is a 62kDa protein. It contains 550 amino acids. The enzyme can be divide into two domains. On the one hand, the major portion, corresponding to the <scene name='60/604470/N_terminal_domain/1'>N-terminal</scene>. On the other hand, the small portion, corresponding to the <scene name='60/604470/C_terminal_domain/1'>C-terminal domain</scene>.Those two domains are separated by a large cleft.

====The C terminal domain====
The amino acids of C terminal sequence is composed of contiguous residues and form a type of lid upon the N terminal domain. It contains two β sheets: the <scene name='60/604470/Beta_sheet/2'>first β sheet </scene> is composed of two short antiparallel strands and the <scene name='60/604470/Beta_sheet_2/1'> second β sheet</scene> is composed of 3 antiparallel strands, mixed with <scene name='60/604470/3_alpha_helices/1'>three α helices</scene>. Those helices are put toward the outside.It is α + β structure.

====The N terminal domain====
The amino acids of C terminal sequence is composed of non contiguous residues. It falls into three subdomains (<scene name='60/604470/Domain_a/1'>the subdomain A</scene>,<scene name='60/604470/Domain_b/1'>the subdomain B</scene>, and <scene name='60/604470/Domain_c/1'>the subdomain C</scene>).It has an antiparallel β barrel, two β sheets, which are framed by α helices. Each of the two β sheets subdomains (A and B)are composed of 8 β strands and 6 helices. The β sheet A has 5 parallel and 3 antiparallel β strands. The β sheet B has 6 parallel and 2 antiparallel β strands. Those β sheets create a groove, closed on one end by the β barrel.

== Structure related to functions ==

The most known reaction of luciferase is the light emission where luciferase uses luciferin, ATP and O2 as substrates. The color of emitted light varies according to the pH, which can be explained by the luciferase structure. But there are some other reactions which can uses fatty acids and coenzyme A. So the active site of the luciferase can theorically bind all these compounds.

====Interactions with ligands====

The active site is not strictly highlighted according to the actual state of studies but some residues and motifs strongly modified have been determined and this conformation enables to find the active site. Many of these conserved residue are located on the core of the β-barrel and on the small C-terminal domain and in the surface of the N-terminal domain, which forms a <scene name='60/604470/Depression_c-ter_and_n-ter/1'>depression</scene>. However, this depression is too large to enable interactions between residues and substrates, so it is thought that a conformational change occurs and sandwiches the substrates, forming the active site. This conformational change provide a suitable environment for light production because of water molecules will be excluded from the active site, favouring intramolecular reactions. Residues also follow a <scene name='60/604470/Cleft/1'>cleft</scene> caused by of the <scene name='60/604470/Beta_sheet_b/3'>Beta-sheet B</scene> against the <scene name='60/604470/Beta_barrel/3'>Beta-barrel</scene>.<ref>PMID:8805533</ref>

[[Image:Luciferin_bounding_to_luciferase.jpg|250px|right|thumb|Hydrogen bonding between Luciferase and substrates luciferin (green), ATP (violet) and Mg2+,<ref>[http://www.photobiology.info/ Photobiology]</ref>]]
=====Interaction with ATP=====
We find a signal motif in luciferase which is <scene name='60/604470/Atp_binding_signal_motif/1'>[STG]-[STG]-G-[ST]-[ST]-[TSE]-[GS]-x-[PALIVM]-K</scene> where some residue like lysine are always conserved. This pattern enables ATP binding thanks to hydrogen bonds between residues and phosphates of ATP. There is another pattern : <scene name='60/604470/Adenosine_ring_binding/1'>[YFW]-[GASW]-x-[TSA]-E</scene> which takes a particular conformation because of hydrogen bonds between residues and maintain the adenosin ring of ATP.<ref>PMID:8805533</ref>, <ref>[http://www.photobiology.info/ Photobiology]</ref>
=====Interaction with luciferin=====
Luciferase holds the luciferin with the specific residues <scene name='60/604470/Residues_helding_luciferin/1'>arginin 218, phenylalanin 247, serin 347 and adenin 348</scene>, still with hydrogen bounds. This bindings makes the carboxylate oxygen of luciferin points toward the α phosphate of ATP, so the oxygen is well-positionned to attack the α phosphate. This promotes the luciferin-AMP formation.<ref>PMID:8805533</ref>, <ref>[http://www.photobiology.info/ Photobiology]</ref>
=====Interaction with fatty acids=====
Fatty acids are highly similar to luciferin. Therefore, luciferase can use the luciferin binding site to bind fatty acids. That is why they can be used as substrates by luciferase and then, very high similar reaction as for luciferin occurs.
[[Image:Luciferin_fatty-acid.gif|250px|left|thumb|Comparison of the chemical structures of (A) firefly
D-LH2 and (B) arachidonic acid,<ref>PMID:18949818</ref>]]

====Color modulation====

When the pH is low, the color of light changes. This is probably due to the hydrogen bonds network between substrates, residues of the cleft and water. Indeed, this network triggers to an external electrostatic potential which stabilizes a charge created during the reaction of light emission. The lower pH leads to a weakening of the network, so the energy which can be emitted decreases and the wavelength of photon increases and becomes more red.<ref>PMID:21188462</ref>

== Evolution ==

The number of proteins related to luciferase is growing exponentially. They are a lot of enzymes very different involved in a lot of mechanisms such as biosynthesis of siderophores, of antibiotics, fatty acid:coenzyme A ligase, and so on...
All the coenzyme A ligase show a very high level of similarity : indeed, each of these enzyme related to luciferase catalyze the adenylation of a carboxylic acid substrate using ATP-Mg2+ and then, the ligation of the activated carboxylic acid with an acceptor.
That is why luciferase is more and more considered as coming from a common ancestor involved in this kind of reaction and the light production would only be a side effect of the reaction.<ref>PMID:19859663</ref>

</StructureSection>
== References ==
<references/>

2015-01-08T21:06:50Z

Amandine Schmit:

<Structure load='1LCI' size='350' frame='true' align='right' caption='Crystal structure of firefly luciferase' scene='Insert optional scene name here' />

==Biological role==
Firefly, also named Photinus pyralis, is a bioluminescent insect. They are able to produce light by a very energetic process, in order to attract its mate. The enzyme responsible of this light producing is luciferase, also known as luciferin-4-monooxygenase (EC: 1.13.12.7). It has various applications in the biotechnology field. In fact, it is used in chemical biology and drug assays. For example, they help to detect protein-protein interactions, to track cells in vivo in order to analysis in real-time the development of a disease in molecular level, to monitor the transcriptional and post-transcriptional regulation of specifics gens, to control apoptosis, to label cancer cells, to detect environmental contamination, to follow protein trafficking, … As there is no light production by mammals, luciferase is a really a great tool for researchers. But this light emission depends on the environmental conditions. Luciferase acts also as a ligase.
Thus, this oxydo-reductase is involved in severals reactions.
=====Light emission=====
The most known reaction of luciferase is the light emission. In this reaction, luciferase firstly synthetized luciferin-AMP from luciferin and ATP, using a Mg2+ ion to offset the negative charges of the phosphate groups. Then, luciferase turns the luciferin-AMP into oxyluciferin in an excited state thanks to a dioxygen. This step releases AMP and CO2. The excited oxyluciferin relaxes and looses a photon so light is emitted.
The wavelength of the light can vary with the pH : at the physiological pH, the emitted light is green and at a lower pH, the color is red.
=====Fatty-acyl-CoA synthesis=====
This reaction uses similar reaction but instead of taking luciferin as ligand, it takes fatty acids. Using ATP-Mg2+, it firstly forms fatty-acyl-AMP. And then, CoA-SH attacks the carboxylic group of fatty-acyl-AMP and so forms fatty-acyl-CoA.
=====Luciferin & Coenzyme A=====
It has been found that the reaction between luciferin and CoA is possible, forming in a first step luciferin-AMP and then luciferin-CoA. This reaction leads to an interesting biological phenomenon : when this reaction occurs in parallel of light emission reaction, we don't have a flash of light but a continuous light emission. This is because luciferin-AMP is a competitive inhibitor of the reaction whereas the luciferin-CoA is not. So the inhibition is deleted and the reaction continue to occur.

== Global Structure ==
Luciferase is a 62kDa protein. It contains 550 amino acids. The enzyme can be divide into two domains. On the one hand, the major portion, corresponding to the N-terminal. On the other hand, the small portion, corresponding to the C-terminal. Those two domains are separated by a large cleft.

===The C terminal domain===
The amino acids of C terminal sequence is composed of contiguous residues and form a type of lid upon the N terminal domain. It contains two β sheets: one is composed of two short antiparallel strands and the other one is composed of 3 antiparallel strands, mixed with 3 α helices. Those helices are put toward the outside.It is α + β structure.

===The N termainal domain===
The amino acids of C terminal sequence is composed of non contiguous residues. It falls into three subdomains (noted A, B, and C).It has an antiparallel β barrel, two β sheets, which are framed by α helices. Each of the two β sheets subdomains (A and B)are composed of 8 β strands and 6 helices. The β sheet A has 5 parallel and 3 antiparallel β strands. The β sheet B has 6 parallel and 2 antiparallel β strands. Those β sheets create a groove, closed on one end by the β barrel.

== Structure related to functions ==

The most known reaction of luciferase is the light emission where luciferase uses luciferin, ATP and O2 as substrates. The color of emitted light varies according to the pH, which can be explained by the luciferase structure. But there are some other reactions which can uses fatty acids and coenzyme A. So the active site of the luciferase can theorically bind all these compounds.

===Interactions with ligands===

The active site is not strictly highlighted according to the actual state of studies but some residues and motifs strongly modified have been determined and this conformation enables to find the active site. Many of these conserved residue are located on the core of the β-barrel and on the small C-terminal domain and in the surface of the N-terminal domain, which forms a <scene name='60/604470/Depression_c-ter_and_n-ter/1'>depression</scene>. However, this depression is too large to enable interactions between residues and substrates, so it is thought that a conformational change occurs and sandwiches the substrates, forming the active site. This conformational change provide a suitable environment for light production because of water molecules will be excluded from the active site, favouring intramolecular reactions. Residues also follow a <scene name='60/604470/Cleft/1'>cleft</scene> caused by of the <scene name='60/604470/Beta_sheet_b/2'>β-sheet</scene> against the <scene name='60/604470/Beta_barrel/2'>β barrel</scene>.

=====Interaction with ATP=====
We find a signal motif in luciferase which is <scene name='60/604470/Atp_binding_signal_motif/1'>[STG]-[STG]-G-[ST]-[ST]-[TSE]-[GS]-x-[PALIVM]-K</scene> where some residue like lysine are always conserved. This pattern enables ATP binding thanks to hydrogen bonds between residues and phosphates of ATP. There is another pattern : <scene name='60/604470/Adenosine_ring_binding/1'>[YFW]-[GASW]-x-[TSA]-E</scene> which takes a particular conformation because of hydrogen bonds between residues and maintain the adenosin ring of ATP.
=====Interaction with luciferin=====
Luciferase holds the luciferin with the specific residues arginin 218, phenylalanin 247, serin 347 and adenin 348, still with hydrogen bounds. This bindings makes the carboxylate oxygen of luciferin points toward the α phosphate of ATP, so the oxygen is well-positionned to attack the α phosphate. This promotes the luciferin-AMP formation.
=====Interaction with fatty acids=====
Fatty acids are highly similar to luciferin. Therefore, luciferase can use the luciferin binding site to bind fatty acids. That is why they can be used as substrates by luciferase and then, very high similar reaction as for luciferin occurs.

===Color modulation===

When the pH is low, the color of light changes. This is probably due to the hydrogen bonds network between substrates, residues of the cleft and water. Indeed, this network triggers to an external electrostatic potential which stabilizes a charge created during the reaction of light emission. The lower pH leads to a weakening of the network, so the energy which can be emitted decreases and the wavelength of photon increases and becomes more red.

== Evolution ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 951

2015-01-08T20:39:42Z

Amandine Schmit:

2015-01-07T22:50:42Z

Amandine Schmit:

Sandbox Reserved 951

2015-01-07T22:49:28Z

Amandine Schmit:

===Firefly Luciferase===

<Structure load='1LCI' size='350' frame='true' align='right' caption='Crystal structure of firefly luciferase' scene='Insert optional scene name here' />

== Introduction==

==Biological role==

== Global Structure ==

== Function highlighted with structure ==

== Evolution ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 951

2015-01-07T22:47:46Z

Amandine Schmit:

==='''Firefly Luciferase'''===

<Structure load='1LCI' size='350' frame='true' align='right' caption='Crystal structure of firefly luciferase' scene='Insert optional scene name here' />

== Introduction==

==Biological role==

== Global Structure ==

== Function highlighted with structure ==

== Evolution ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>

Sandbox Reserved 951

2015-01-07T22:44:00Z

Amandine Schmit:

==='''Firefly Luciferase'''===

<Structure load='1LCI' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

== Introduction==

==Biological role==

== Global Structure ==

== Function highlighted with structure ==

== Evolution ==

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

</StructureSection>
== References ==
<references/>