Japanese firefly Luciferase complexed with Oxyluciferin & AMP
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| 2d1r, resolution 1.60Å (default scene) | |||||||||||||
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| Ligands: | AMP, OLU | ||||||||||||
| Non-Standard Residues: | CSO | ||||||||||||
| Activity: | Photinus-luciferin 4-monooxygenase (ATP-hydrolyzing), with EC number 1.13.12.7 | ||||||||||||
| Related: | 2d1s, 2d1t | ||||||||||||
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| Resources: | FirstGlance, OCA, RCSB, PDBsum, TOPSAN | ||||||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||||||
Crystal structure of the thermostable Japanese firefly Luciferase complexed with OXYLUCIFERIN and AMP
Introduction
Luciferase is a class of enzymes producing light through the process of bioluminescence. This luciferase was extracted from Luciola cruciata. In the case of fireflies, it catalyses a reaction of adenylation and then an oxydative decarboxylation, changing luciferin to oxyluciferin and thus emitting light. This protein is constituted of two main domains separated by a cleft.
The structure
2D1R is a 1 chain structure of sequence from Luciola cruciata. Full crystallographic information is available from OCA. The protein is shown here bound with two products, adenosine 5'-phosphate (AMP) and oxyluciferine.
It weights 62 kDa and is constituted of two main domains, a large N-terminal domain and a small C-terminal domain, separated by a cleft. The N-ter domain (residues 1-436) is made of a β-barrel and two β-sheets, flanked by α-helices, thus forming an αβαβα five-layered structure. The C-ter domain (residues 440-550) forms a small α+ β domain.
There are two conformational states, one when the enzyme is substrate-free, and the other one product-bound (whose structure is represented on this page). The transition is achieved by a 90° counterclockwise rotation of the C-domain relative to the N-domain
Sequence similarities
The firefly luciferase shares homologuous sequences and mechanisms with the peptide synthetases and acyl-CoA ligases, with a very few residues always conserved. The firefly luciferase belongs to the super-family of acyl-adenylate-forming and thioester-forming enzymes.
But the firefly luciferase also have caracteristic sequences found in no other nucleotide-binding proteins, such as a motif responsible for the binding of ATP.
Activation site
The firefly luciferase interacts with ATP, luciferin, an Acetyl-coenzyme A (CoA )and O2. So differents groups of residues are part of the the ligand binding. Those residues can be placed in the molecule by comparing the sequences of different enzymes of the same family. The ATP binds to a 206 G-x-x-x-x-x-G-K-[STG]196 sequence which is a very disordered version of a classical mononucleotide-binding motif. (Réf à mettre) The luciferin-binding site seems to be a depression between β sheet B and the barrel subdomain, but is not clearly visible on the structure. The CoA-binding site does not have a classical motif of adenylate forming enzyme and this luciferase is he first member of a new superfamily to be studied. The adenylate forming reaction is likely to involve a few glycine or charged residues. The others binding sites are not clearly defined but here are the conserved residues taking part in the binding or the catalysis:
• Thr 346 which is the only residue with dihedral angles outside of the usual regions of the Ramachandran plot. This energetically unfavorable angles are usually in relation with residues having important functional roles.
• Asp 422 is an invariant residue exposed to the solvant, and could make an hydrogen bond with the side chain of another well-conserved residue, the Tyr340.
• The Ser420 has an hydroxyl group able to make hydrogen bond with the Asp422 and gly421 which are close enough.
All those conserved residues are either on the surface of the two domains on the side of the cleft or on the loop connecting the two domains. (lien vert sur 'conserved residues' qui les place sur la molécule ?) But this cleft is too big to accomodate on the substrates and is seems tha the two domains only come closer around the products when they are formed.
Applications of the luciferase
Luciferase can be used to measure ATP Cite error: Closing </ref> missing for <ref> tag
It can also be used to study the action of general anesthetics[1], which are inhibiting it.
The applications for luciferase are very diverse.
Chemical reaction
The enzyme catalyses the production of light, using two different conformation to catalyze two half-reactions (in the case of firefly Luciferase).
- luciferin + ATP → luciferyl adenylate + PPi
- luciferyl adenylate + O2 → oxyluciferin + AMP + light
The first half-reaction is an adenylation, the second one is an oxydative decarboxylation.
The production of light is achieved by the conversion of chemical energy into an emission of photons, resulting from the passage of the oxyluciferin's excitation state to a ground state.
Spectral difference with mutated luciferases
The colour changes from the classical yellow-green colour to red with the substitution of a single amino acid, Ser 286 to Asn, in the S286N mutant. The active site (and the residue Ile 288) is less potent to effectue it's conformational change to the closed state, which was providing an extremely hydrophobic environment. This is then allowing an energy loss, and the product will emit lower energy light, with a wavelength moved to the red. Further mutations proved that the amino acid residue at position 288 is influencing the wavelength of the light emitted.[2]
Biology
The bioluminescent systems in the living organisms are very diverse (for example the luciferase has a different structure and catalyses a reaction in one step in the bacteria), so it is thought that they appeared separately in the course of evolution. The fireflies use bioluminescence to locate other indivuduals for mating, or to lure other species which are their preys. In the larvae, it is a warning signal for the predators, implying the presence of toxins.
References
- Elena Conti, Nick P Franks, Peter Brick. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. Volume 4, Issue 3, March 1996, Pages 287-298 doi:10.1016/S0969-2126(96)00033-0
- Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence. Nature. 2006 Mar 16;440(7082):372-6. PMID:16541080 doi:10.1038/nature04542
- Szarecka A, Xu Y, Tang P. Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses. Biophys J. 2007 Sep 15;93(6):1895-905. Epub 2007 May 18. PMID:17513367 doi:10.1529/biophysj.106.102780
- Hawronskyj J.-M, Measurement of ATP using firefly luminescence. European food and drink review. 1997, SUMMER, pp. 61-63 ISSN 0955-4416
- ↑ Szarecka A, Xu Y, Tang P. Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses. Biophys J. 2007 Sep 15;93(6):1895-905. Epub 2007 May 18. PMID:17513367 doi:10.1529/biophysj.106.102780
- ↑ Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for the spectral difference in luciferase bioluminescence. Nature. 2006 Mar 16;440(7082):372-6. PMID:16541080 doi:10.1038/nature04542
