Renilla-luciferin 2-monooxygenase

 

Oblein, a protein from the bio-luminescent hydroid, Obleia longissima, is a calcium-regulated photoprotein. The addition of calcium regulates, but is not necessary for bio-luminescence and there is no requirement for an additional substrate (e.g. molecular oxygen). Calcium-regulated photoproteins are found in and are responsible for the light emission of a variety of bio-luminescent marine organisms, mostly coelenterates. Obelin consists of a single polypeptide chain to which the substrate, 2-hydroxy-coelenterazine is tightly, but not covalently bound. Calcium binding results in blue bio-luminescence via oxidative carboxylation of the substrate to the excited state coelenteramide product through several intermediates.

 

Reference Protein and Structure

Sequence
Q27709 UniProt IPR011992 (Sequence Homologues) (PDB Homologues)
Biological species
Obelia longissima (Black sea hydrozoan) Uniprot
PDB
1qv0 - Atomic resolution structure of obelin from Obelia longissima (1.1 Å) PDBe PDBsum 1qv0
Catalytic CATH Domains
1.10.238.10 CATHdb (see all for 1qv0)
Cofactors
Calcium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:1.13.12.5)

Renilla luciferin
CHEBI:16531ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
oxidized Renilla luciferin
CHEBI:17959ChEBI
+
photon
CHEBI:30212ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
Alternative enzyme names: Renilla-type luciferase, Aequorin, Luciferase (Renilla luciferin), Luciferase, Renilla-luciferin 2-monooxygenase, Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating),

Enzyme Mechanism

Introduction

The substrate, 2-hydroxy-coelenterazine is tightly, but not covalently bound to the oblein active site. Calcium binding initiates oxidative decarboxylation of the substrate to the product, coelenteramide, in its excited form. This proceeds via several intermediates with no requirment for added molecular oxygen since this is derived from the peroxy substitution of the coelenterazine itself. The energy is deposited into the excited state of the product, followed by the emission of a photon.

Metal binding changes the His 175-Tyr 190 distance. The His 175 acts as a general base, and deprotonates the Tyr 190, which is hydrogen bonded to the substrate. The Tyr 190 then acts as a base on the substrate deprotonating it to leave an amide anion, stabilised by hydrogen bonding to Tyr 138. Decarboxylation occurs with a rearrangement, forming the amide ion excited state product.

Proton transfer to His 22 generates the excited phenolate ion. This is stabilised by a His 22-phenolate ion pair, and also by the side group amine of Trp 92. Formation of the product from this stabilised excited form, with the emission of a photon (bioluminescence).

Catalytic Residues Roles

UniProt PDB* (1qv0)
His22 His22A The His 22 imidazole ring is hydrogen bonded to the phenol group of the substrate. His 22 deprotonates the hydroxyl group, leaving a phenolate anion, stabilised by the His 22-phenolate ion pair interaction. proton acceptor, electrostatic stabiliser
Trp92 Trp92A The amine side chain of Trp 92 stabilises the phenolate-His 22 ion pair via a hydrogen bond. electrostatic stabiliser
Tyr138 Tyr138A Tyr 138 stabilises the amide anion via a hydrogen bond. electrostatic stabiliser
His175 His175A His 175 acts as a general base, deprotonating the Tyr 190, activating the Tyr residue to deprotonate the substrate. proton acceptor, electrostatic stabiliser
Tyr190 Tyr190A The Tyr 190 acts as a base on the substrate, depronating it to form the amide anion. proton relay, proton acceptor, electrostatic stabiliser, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

aromatic bimolecular nucleophilic addition, intermediate formation, overall reactant used, proton transfer, bimolecular nucleophilic addition, decarboxylation, intermediate collapse, overall product formed, proton relay

References

  1. Vysotski ES et al. (2004), Acc Chem Res, 37, 405-415. Ca2+-Regulated Photoproteins:  Structural Insight into the Bioluminescence Mechanism. DOI:10.1021/ar0400037. PMID:15196050.
  2. Malikova NP et al. (2003), FEBS Lett, 554, 184-188. Spectral tuning of obelin bioluminescence by mutations of Trp92. DOI:10.1016/s0014-5793(03)01166-9. PMID:14596937.

Step 1. The first step in catalysis is the hydroperoxidation of coelenterazine to form a coelenterazine 2-hydroperoxide intermediate.

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Catalytic Residues Roles

Residue Roles
Tyr138A electrostatic stabiliser
His22A electrostatic stabiliser
Trp92A electrostatic stabiliser
His175A electrostatic stabiliser
Tyr190A electrostatic stabiliser

Chemical Components

ingold: aromatic bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 2. Ca2+ binding causes the Tyr190-His175 hydrogen bon to become stronger. His175 acts as a base to deprotonate Tyr190. The hydroperoxide protonatesd the tyrosinate, forming a peroxy anion.

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Catalytic Residues Roles

Residue Roles
His22A electrostatic stabiliser
Trp92A electrostatic stabiliser
Tyr138A electrostatic stabiliser
His175A proton acceptor
Tyr190A proton donor, proton relay, proton acceptor

Chemical Components

proton transfer

Step 3. There is irreversible nucleophilic attack of the peroxy anion on the C3-carbon of coelenterazine to form a dioxetanone intermediate.

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Catalytic Residues Roles

Residue Roles
His22A electrostatic stabiliser
Trp92A electrostatic stabiliser
Tyr138A electrostatic stabiliser
His175A electrostatic stabiliser
Tyr190A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition

Step 4. The dioxetanone intermediate collapses and carbon dioxide is released.

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Catalytic Residues Roles

Residue Roles
His22A electrostatic stabiliser
Trp92A electrostatic stabiliser
Tyr138A electrostatic stabiliser
His175A electrostatic stabiliser
Tyr190A electrostatic stabiliser

Chemical Components

decarboxylation, intermediate collapse

Step 5. The oxyanion collapses to form the amide anion excited state of the product.

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Catalytic Residues Roles

Residue Roles
His22A electrostatic stabiliser
Trp92A electrostatic stabiliser
Tyr138A electrostatic stabiliser
Tyr190A electrostatic stabiliser

Chemical Components

overall product formed

Step 6. Subsequent excited state proton transfer is proposed to transform the product into the excited phenolate ion-pair responsible for bioluminescence emission at longer wavelength. His22 acts as a base to deprotonate the phenol group. The product and native state of the enzyme can be restored by reversible proton transfer.

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Catalytic Residues Roles

Residue Roles
Trp92A electrostatic stabiliser
Tyr138A electrostatic stabiliser
His175A electrostatic stabiliser
Tyr190A electrostatic stabiliser
His22A proton acceptor

Chemical Components

proton relay
Download: Image, Marvin File

Step 1. The first step in catalysis is the hydroperoxidation of coelenterazine to form a coelenterazine 2-hydroperoxide intermediate.

Step 2. Ca2+ binding causes the Tyr190-His175 hydrogen bon to become stronger. His175 acts as a base to deprotonate Tyr190. The hydroperoxide protonatesd the tyrosinate, forming a peroxy anion.

Step 3. There is irreversible nucleophilic attack of the peroxy anion on the C3-carbon of coelenterazine to form a dioxetanone intermediate.

Step 4. The dioxetanone intermediate collapses and carbon dioxide is released.

Step 5. The oxyanion collapses to form the amide anion excited state of the product.

Step 6. Subsequent excited state proton transfer is proposed to transform the product into the excited phenolate ion-pair responsible for bioluminescence emission at longer wavelength. His22 acts as a base to deprotonate the phenol group. The product and native state of the enzyme can be restored by reversible proton transfer.

Products.

Contributors

Emma Penn, Gemma L. Holliday, Amelia Brasnett