Renilla-luciferin 2-monooxygenase, Renilla luciferase, or RLuc, is a
bioluminescent
Bioluminescence is the production and emission of light by living organisms. It is a form of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some b ...
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
found in ''
Renilla reniformis
The sea pansy, ''Renilla reniformis,'' is a species of colonial cnidarian in the family Renillidae, part of an octocoral subclass of Anthozoa that inhabit an expansive range of environments. It is native to warm continental shelf waters of th ...
'', belonging to a group of
coelenterazine luciferase
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'' ...
s. Of this group of enzymes, the luciferase from ''
Renilla reniformis
The sea pansy, ''Renilla reniformis,'' is a species of colonial cnidarian in the family Renillidae, part of an octocoral subclass of Anthozoa that inhabit an expansive range of environments. It is native to warm continental shelf waters of th ...
'' has been the most extensively studied, and due to its bioluminescence requiring only molecular oxygen, has a wide range of applications, with uses as a reporter gene probe in cell culture, in vivo imaging, and various other areas of biological research.
Recently, chimeras of RLuc have been developed and demonstrated to be the brightest luminescent proteins to date, and have proved effective in both noninvasive single-cell and whole body imaging.
Note that the EC record also includes other unrelated enzymes that catalyze the same reaction. An example is the calcium-dependent
photoprotein aequorin: while Rluc is in the
AB hydrolase
The alpha/beta hydrolase superfamily is a Protein superfamily, superfamily of hydrolytic enzymes of widely differing phylogenetic origin and catalytic function that share a common Protein fold, fold. The core of each enzyme is an alpha/beta-sheet ...
superfamily, aequorin is an
EF hand
The EF hand is a helix–loop–helix structural domain or ''motif'' found in a large family of calcium-binding proteins.
The EF-hand motif contains a helix–loop–helix topology, much like the spread thumb and forefinger of the human hand, in ...
protein. The name does not specifically refer to ''Renilla'', but instead refers to Renilla-luciferin, a chemical also known as coelenterazine.
Chemical reaction
RLuc is an
oxidoreductase
In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. This group of enzymes usually ...
, specifically acting on single donors with O
2 as the oxidant. However, this enzyme appears to be unrelated from most other luciferases that act on coelenterazine, such as those from
copepods
Copepods (; meaning "oar-feet") are a group of small crustaceans found in nearly every freshwater and saltwater habitat. Some species are planktonic (inhabiting sea waters), some are benthic (living on the ocean floor), a number of species have p ...
.
RLuc
catalyzes
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
the
chemical reaction
A chemical reaction is a process that leads to the chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the positions of electrons in the forming and break ...
Coelenterazine + O
2 coelenteramide
Coelenteramide is the oxidized product, or oxyluciferin, of the bioluminescent reactions in many marine organisms that use coelenterazine. It was first isolated as a blue fluorescent protein from ''Aequorea victoria'' after the animals were stimul ...
+ CO
2 + hν
In the process, coelenterazine is oxidized with a concurrent loss of
CO2, and a
photon
A photon () is an elementary particle that is a quantum of the electromagnetic field, including electromagnetic radiation such as light and radio waves, and the force carrier for the electromagnetic force. Photons are Massless particle, massless ...
of blue light is emitted.
Biological function
In ''Renilla reniformis'', RLuc is found in membrane-bound intracellular structures within specialized light emitting cells, and is coupled with a closely interacting
green fluorescent protein
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aeq ...
(RrGFP), and a Ca
++ activated luciferin binding protein (RrLBP).
Although the luciferase catalyzed oxidation of coelenterazine releases a photon of blue light (480 nm), this is not observed ''in vivo''. Instead, the energy released by the reaction involving RLuc is passed via resonance energy transfer to the fluorophore of RrGFP and emitted as a green photon (505 nm), resulting in green bioluminescence observed from the animal. This process relies on a
Förster resonance energy transfer
Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). ...
(FRET) mechanism, increasing the emitted photon number approximately six-fold.
Structure
Renilla luciferase contains 311
amino acids,
and is active as a nearly spherical single
polypeptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides ...
chain monomer of 36
kDa, which have a tendency for self-association, forming inactive dimers and trimers. Like other
dehalogenase-superfamily enzymes, it has a characteristic α/β-hydrolase fold sequence at its core and shares the conserved catalytic triad of residues employed by
dehalogenases. In RLuc, the loop containing residues 153 – 163 is structurally flexible, facilitating greater diffusion of solvents into the active site, which contains a
highly-conserved catalytic triad
A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes (e.g. proteases, amidases, esterases, acylases, l ...
consisting of
Aspartic Acid
Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α- amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pr ...
at residue 120,
Glutamic Acid
Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
at residue 144, and
Histidine
Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the ...
at residue 285.
Enzyme pathway
Unlike
photoproteins Photoproteins are a type of enzyme, made of protein, from bioluminescent organisms. They add to the function of the luciferins whose usual light-producing reaction is catalyzed by the enzyme luciferase.
History
The term photoprotein was first used ...
which stably bind
coelenterazine and emit light upon addition of calcium, coelenterazine is normally bound by RrLBP, the luciferin-binding protein. When stimulated, a Ca
2+ ion first interacts with RrLBP, causing it to release coelenterazine.
Coelenterazine is then
oxidized
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a de ...
by RLuc into
coelenteramide
Coelenteramide is the oxidized product, or oxyluciferin, of the bioluminescent reactions in many marine organisms that use coelenterazine. It was first isolated as a blue fluorescent protein from ''Aequorea victoria'' after the animals were stimul ...
, releasing a single
photon
A photon () is an elementary particle that is a quantum of the electromagnetic field, including electromagnetic radiation such as light and radio waves, and the force carrier for the electromagnetic force. Photons are Massless particle, massless ...
of blue light (480 nm) in the process. This photon is captured by the adjacent GFP, releasing a photon of
green light. This pathway is summarized below.
Mechanism
The RLuc mediated
chemical reaction
A chemical reaction is a process that leads to the chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the positions of electrons in the forming and break ...
involves the catalytic degradation of coelenterazine, and proceeds through a 1,2-
dioxetane
A dioxetane or dioxacyclobutane is an organic compound with formula C2O2H4, whose backbone is a four-membered ring of two oxygen atoms and two carbon atoms. There are two isomers:
* 1,2-dioxetane where the oxygen atoms are adjacent.
* 1,3-dioxetan ...
(also called dioxetanone or cyclic peroxide) intermediate. Based on studies using radioactively labelled oxygen species within the RLuc complex, it has been determined that the luciferin carbonyl oxygen is exchanged rapidly with oxygen from water prior to incorporation of an oxygen atom from O
2 via a
dioxetane
A dioxetane or dioxacyclobutane is an organic compound with formula C2O2H4, whose backbone is a four-membered ring of two oxygen atoms and two carbon atoms. There are two isomers:
* 1,2-dioxetane where the oxygen atoms are adjacent.
* 1,3-dioxetan ...
intermediate. The resultant CO
2 also rapidly exchanges its oxygens with those from the surrounding water.
The general mechanism is depicted below.
References
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EC 1.13.12
Enzymes of known structure