Luciferases and Light‐emitting Accessory Proteins: Structural Biology


Creatures that glow have mesmerised people for aeons. During the last few hundred years, scientists have been trying to reveal exactly how organisms produce this light, or bioluminescence. It has been estimated that approximately 30 or more chemically distinct bioluminescence systems have evolved independently. The luciferase enzymes that catalyse bioluminescent reactions use a variety of different structures to produce light. This article looks at the X‐ray crystal structures known for five different types of luciferases: bacterial, dinoflagellate, firefly, and two classes of coelenterates (anthozoan and hydrozoan). The structures of these enzymes reveal details of how they catalyse bioluminescence reactions, and their remarkable diversity of structure, mechanism and substrate specificity. Two accessory fluorescent proteins are also described.

Key Concepts:

  • Luciferases are enzymes that catalyse reactions that produce bioluminescence.

  • Approximately 30 distinct bioluminescent systems have evolved.

  • Bioluminescent reactions all involve oxygenation of a substrate to generate a peroxide intermediate, which then breaks down to give an electronically excited product that emits light.

  • X‐ray crystallographic structures are known for the luciferases in five different bioluminescent systems: bacteria, dinoflagellates, fireflies, hydrozoa and anthozoa.

  • Each of these luciferases has an entirely different molecular structure and catalyzes bioluminescence in a unique way.

Keywords: luciferase; luciferin; bioluminescence; chemiluminescence; antenna proteins; green fluorescent protein; yellow fluorescent protein; photoprotein; bacterial luciferase; firefly luciferase; dinoflagellate luciferase; renilla luciferase; aequorin; obelin; clytin; hydrozoa; anthozoa

Figure 1.

The bacterial bioluminescence reaction scheme.

Figure 10.

Close‐up of the obelin active site in state II, in the absence of Ca2+, with 2‐hydroxycoelenterazine bound (PDB ID code 1EL4). Residues involved in the bioluminescence reaction (red) are labelled.

Figure 11.

Formation of the GFP chromophore.

Figure 12.

Diagram of GFP illustrating its β‐can topology (PDB ID code 1GFL). Inside the β‐can, the three residues that form the emitter (modified from Ser65, Tyr66 and Gly67) are shown in red.

Figure 13.

The Renilla bioluminescence reaction scheme.

Figure 14.

(a) Structure of rLUC, oriented to the active site is at the top of the figure (PDB ID 2PSF). The cap domain is in tan, the α/β‐hydrolase fold domain is in light purple, and active site residues (D120, E144, and H285) are in red. (b and c) Space filling diagrams of rLUC with the gateway facing the reader, demonstrating the two different active site conformations (arrows) so far reported. The wider gateway with coelenteramide bound and a bowl‐shaped active site can be seen in (b) (PDB ID 2PSJ). The narrow gateway with a vase‐shaped active site is pictured in (c) (PDB ID 2PSD).

Figure 15.

(a) rCBP from R. muelleri with coelenterazine (yellow) bound in the centre of the protein (PDB ID 2HPS). The C‐terminal domain is in light purple, the N‐terminal domain is in tan. (b) Detail of residues binding coelenterazine in rCBP. C‐terminal domain residues are coloured light purple, N‐terminal domain are coloured tan. (c) R. renformis GFP (PDB ID 2PSL) with the chromophore in red. Dashed lines between atoms indicate hydrogen bonds or electrostatic interactions.

Figure 16.

The two‐step firefly bioluminescence reaction scheme. Step 1: adenylate‐forming reaction; step 2: oxidation reaction.

Figure 17.

Structures of the two conformations of Photinus pyralis firefly luciferase. The adenylate‐forming conformation (a) and cross‐linked oxidation conformation (b) are both bound to the luciferyl‐adenylate intermediate analogue 5′‐O‐[N‐(dehydroluciferyl)‐sulfamoyl] adenosine (DLSA); details of their respective active sites are pictured below each structure (PDB ID codes 4G36 and 4G37, respectively). The large N‐terminal domain is in light purple and the small C‐terminal domain is in tan. The DLSA molecule (yellow) indicates the binding site of the enzyme. In (a), Ile108 and Tyr447 are in red, with Tyr447 situated on the far side of the C‐terminal domain. In (b), the equivalent mutated residues, Cys108 and Cys447 are also in red, and the rotation of the C‐terminal domain has placed the two residues close enough to form a disulfide bond. Dashed lines between atoms indicate hydrogen bonds or electrostatic interactions.

Figure 2.

(a) Structure of the FMN‐bound bacterial luciferase heterodimer showing α and β subunits (PDB ID: 3FGC). Bound FMN (spheres representation) indicates the binding site. (b) Close‐up of the active site.

Figure 3.

Structure of LumP in complex with the fluorophore DMRL (stick representation) (PDB ID: 3A3G).

Figure 4.

Schematic of scintillons.

Figure 5.

The dinoflagellate bioluminescence reaction scheme.

Figure 6.

Domain organisation of L. polyedrum luciferase (137 kDa).

Figure 7.

The structure of domain 3 of luciferase from Lingulodinium polyedra (PDB ID: 1VPR). The N‐terminal 3‐helix bundle (light purple) sits on top of the central 10‐stranded barrel (tan), which is flanked by helical and coil structures (blue). Histidines involved in pH regulation of enzyme activity (H899, H909, H924, H930) are in red.

Figure 8.

The hydrozoan bioluminescence reaction scheme.

Figure 9.

Conformation states of the photoprotein obelin: state I, apo‐obelin (structure not determined); II, obelin with 2‐hydroperoxycoelenterazine in the absence of Ca2+ (PDB ID code 1EL4) (Liu et al., ); III, Ca2+‐discharged obelin with both coelenteramide and bound Ca2+ (PDB ID code 2F8P) (Liu et al., ); IV, Ca2+‐discharged obelin with coelenteramide but without Ca2+ (PDB ID code 1S36) (Deng et al., ); V, Ca2+‐loaded apo‐obelin (PDB ID code 1SL7) (Deng et al., ). All structures are presented in the same orientation; the carbon atoms of substrates and products are represented as yellow sticks, and calcium ions are represented as green balls. Reproduced by permission of PNAS Figure (p. 2571) from Liu et al. (). © National Academy of Sciences, USA.



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Sharpe, Miriam L, Hastings, J Woodland, and Krause, Kurt L(Apr 2014) Luciferases and Light‐emitting Accessory Proteins: Structural Biology. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003064.pub2]