Bioluminescence, a form of chemiluminescence, is commonly known as the production and emission of light by living organisms. Bioluminescent organisms are found in both terrestrial and aquatic environments and these include bacteria, insects, fungi, marine coelenterates as well as crustacea (1). An example of bioluminescence is shown in figure 1 (courtesy of The Independent) with the radiance of algae in the sea. The biological function of bioluminescence is highly species-dependent and ranges from distracting predators to attracting prey or mating partners. In bioluminescence, light is produced through conversion of chemical energy into an excited electronic state which emits a photon of visible light upon relaxation. It has been estimated that Nature harbors about 30 different bioluminescent systems with different biochemical properties (1,2), suggesting that the ability to produce light arose multiple times during evolution. Typically, bioluminescent organisms employ an enzymatic system, comprising an oxidative enzyme called luciferase and its substrate luciferin for the production of light. Although all luciferases catalyze an oxidation reaction, this is accomplished by utilizing different cofactors, unrelated substrates and specific reaction pathways. For example, bacterial luciferase is a flavin monooxygenase that catalyzes the oxidation of long chain aldehyde (3), while firefly luciferase does not require any cofactor and catalyzes an oxidative decarboxylation. Firefly luciferase has been studied for decades and is used in a variety of molecular and cell biology applications, like the quantification of ATP and reporter for gene expression. The firefly luciferase/luciferin combination is undoubtedly the best characterized bioluminescent system. Here, I will discuss biochemical properties, the structure and mechanistic understanding of firefly luciferase.
Biochemical properties of luciferase
Firefly luciferase is a peroxisomal protein of 62 kDa, residing in the light-emitting organ of the firefly, called the lantern. The enzyme is equipped with a C-terminal peroxisomal targeting peptide (Ser-Lys-Leu), which enables the post-translational import into peroxisomes. Firefly luciferase is classified as a monooxygenase and catalyzes the emission of light (yellow/green) through oxidation of D-luciferin in the presence of ATP and oxygen (2). Luciferase belongs to the ANL superfamily of adenylating enzymes, which also includes acyl-CoA synthetases and adenylation domains of non-ribosomal peptide synthetases (NRPS) (4). Figure 2 (adopted from 4) shows the reaction catalyzed by firefly luciferase. In general, ANL family members catalyze a two-step reaction of which the first one is used to activate a carboxylate substrate through a reaction with ATP, yielding an adenylate intermediate (luciferyl•AMP) and PPi. This intermediate provides the energy for the second half reaction, which is for luciferase an oxidative decarboxylation. This reaction liberates AMP and generates a second intermediate (oxyluciferin) that decomposes within the enzyme, liberating a photon of light (4). The initial reaction of acyl-CoA synthetases and adenylation domains comprises the formation of an acyl-adenylate. Subsequently, the acyl moiety is transferred to the thiol group of CoA or pantetheine through a thioester-forming reaction (4). Interestingly, firefly luciferase is also able to bind CoA. In fact, it has been established that firefly luciferase is active with long-chain unsaturated fatty acids in the presence of ATP and CoA, resulting in the formation of fatty-acyl CoA derivatives (2) similar to acyl-CoA synthetases. Hence, luciferase is a bifunctional enzyme capable of catalyzing the bioluminescence reaction as well as fatty acyl-CoA synthesis.
Structure of luciferase
The primary sequence of firefly luciferase consists of 550 residues in a single polypetide chain and contains the characteristic motifs of ANL family members, comprising phosphate and nucleotide-binding sites. The first high resolution structure of firefly luciferase was solved in 1996 at a resolution of (5). This structure (PDB 1LCI) is shown in figure 3 and reveals that the enzyme is folded in two domains, namely a large N-terminal domain (in magenta) and a small C-terminal domain (2Å in green). The N-terminal domain is made up of an antiparallel β-barrel and two β sheets, which are flanked by α-helices, while the C-terminus comprises a typical α/β domain, made up of parallel β-sheets surrounded by α-helices. However, this structure was solved in the absence of ligands and does therefore not provide detailed information on the location of the active site. Therefore, the structure of luciferase from the Japanese firefly bound several ligands, including an analogue of the natural luciferyl adenylate (DLSA), was determined at a resolution of 1.3Å (6). This structure is shown in figure 3 (PDB 2D1S) and demonstrates that its overall structure is similar to the structure of firefly luciferase with a large N-terminal domain (magenta) and small C-terminal domain (green). Both domains are joined via a small flexible linker. DLSA (in blue and shown as spheres) is sandwiched between domains with the luciferin-binding site located at the N-terminal domain (6).
Recent structural evidence established that acyl-CoA synthetases and NRPS adenylation domains employ a remarkable catalytic strategy, involving a rotation of the C-terminal domain by 140o. This domain alteration enables the use of a single active site for the catalysis of two half reactions (4). Specifically, the C-terminal domain rotates following formation of the acyl-adenylate, this allows CoA or pantetheine to enter the active site through the so-called pantetheine tunnel allowing the carbonyl of the adenylate intermediate to be attacked by the thiol of CoA or pantetheine in a thioester-forming reaction.
Firefly luciferase is a member of the same protein family as acyl-CoA synthetases and adenylation domains and it can therefore be expected that luciferase employs a similar catalytic strategy as other ANL family members. To confirm whether a domain alteration is also observed during the catalytic cycle of luciferase, the structure of luciferase from the eastern firefly was solved in two conformations. To trap the enzyme in the adenylate-forming conformation, it was bound to DLSA, an analogue of luciferyl adenylate. Subsequently, its structure was solved at a resolution of 2.6Å (7). This structure (PDB 4G36) is shown in figure 4 and reveals that the enzyme is made up of a large N-terminal domain (magenta) and a small C-terminal domain (green) like other luciferases. DLSA (in blue spheres) is located at the interface between both domains with the active site localized to the N-terminal domain. To capture the enzyme in the second conformation, eastern firefly luciferase bound to DLSA was treated with a chemical cross-linker Subsequently, its crystal structure was solved at a resolution of 2.4Å (7). This structure (PDB 4G37) is displayed in figure 4 with the N-terminal domain in red and the C-terminal domain in yellow. DLSA is shown in orange spheres. A structural alignment of both conformations reveals a rotation of the C-terminal domain in the structure poised to catalyze the second step as has been observed in acyl-CoA synthetases and adenylation domains. Therefore, luciferase employs a similar catalytic strategy as other ANLfamily members. Rotation of the C-terminal domain creates the pantetheine tunnel in acyl-CoA synthetases and adenylation, allowing CoA or pantetheine to enter the active site. This tunnel is also present in the structure of luciferase poised to catalyze the second reaction. Interestingly, a substitution of a conserved residue that lines the tunnel in luciferase impairs the oxidative decarboxylation, suggesting that the pantetheine tunnel is used by oxygen to enter the active site (7).
Although the precise light-emitting species in firefly bioluminescence has not been conclusively identified, it is generally accepted that luciferin adenylate is oxygenated with molecular oxygen, yielding the excited state of oxyluciferin, AMP and CO2. Subsequently, a photon of light is emitted upon relaxation of excited oxyluciferin to the ground state (figure 5 adopted from 6) (2,7).
Chemiluminescence is known as the emission of light through a chemical reaction and this process also occurs in living organisms. In this case it is called bioluminescence, which is in fact a widely occurring phenomenon in Nature. Light-emitting organisms are found in terrestrial and aquatic environments. Notable examples include the glow of bacteria on decaying meat or fish, the radiance of algae in the sea (figure 1), or the flickering signals of fireflies. Bioluminescence has distinct biological functions in different organisms. For instance, it is used as camouflage by certain squids, attraction of mates by fireflies, defense against predators by crustaceans, as warning signal by jelly fish and as communication (quorum sensing) by bacteria. Remarkably, Nature harbors about 30 different bioluminescent systems with different biochemical properties (1,2), suggesting that the ability to produce light arose multiple times during evolution. In bioluminescence, light is produced through conversion of chemical energy into an excited electronic state which emits a photon of visible light upon relaxation. It has been established that all light-emitting organisms employ an enzymatic system for the production of light, comprising an oxidative enzyme called luciferase and its substrate luciferin. Despite the general principles of the bioluminescent reaction, the nature of the enzymes involved differs substantially. For example, bacterial luciferase is a flavin monooxygenase that catalyzes the oxidation of longchain aldehyde (3), while firefly luciferase does not require any cofactor and catalyzes an oxidative decarboxylation (2). Owing to its unique properties (high quantum yield and large dynamic range), luciferase has emerged as a popular enzyme in molecular biology applications, ranging from ATP quantification to reporter for gene expression and high-throughput drug discovery (1,8). The detailed structural information of firefly luciferase and related enzymes bound to ligands and in different catalytic conformations has furthered the mechanistic understanding of these enzymes at the molecular level, enabling the engineering of luciferase to obtain variants emitting non-natural light (6) as well as improved versions for bioanalytic purposes (9). Moreover, the detailed understanding of luciferase conferred bioluminescence into fatty acyl-CoA synthases from non-bioluminescent beetles (2).
1. Kaskova ZM, Tsarkova AS, Yampolsky IV. 2016. 1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine. Chem Soc Rev. 45 pp: 6048-6077.
2. Inouye S. 2010. Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions. Cell Mol Life Sci. 67 pp :387-404.
3. Tinikul R, Chaiyen P. 2016. Structure, Mechanism, and Mutation of Bacterial Luciferase. Adv Biochem Eng Biotechnol. 154 pp: 47-74.
4. Gulick AM. 2009. Conformational dynamics in the Acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem Biol. 4 pp: 811-827.
5. Conti E, Franks NP, Brick P. 1996. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 4 pp: 287-298.
6. Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. 2006. Structural basis for the spectral difference in luciferase bioluminescence. Nature. 440 pp: 372-376.
7. Sundlov JA, Fontaine DM, Southworth TL, Branchini BR, Gulick AM. 2012. Crystal structure of firefly luciferase in a second catalytic conformation supports a domain alternation mechanism. Biochemistry. 51 pp: 6493-6495.
8. Scott D, Dikici E, Ensor M, Daunert S. 2011. Bioluminescence and its impact on bioanalysis. Annu Rev Anal Chem. 4 pp: 297-319.
9. Branchini BR, Ablamsky DM, Murtiashaw MH, Uzasci L, Fraga H, Southworth TL. 2007. Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem. 361 pp: 253-262.