Luciferase (P. pyralis)
Created by Chao Zhang
Bioluminescence in nature is carried out by the conversion of chemical energy into light. Such processes are achieved by the chemical oxidation of a luciferin substrate catalyzed by a luciferase enzyme. The firefly luciferase (PDB ID: 1BA3), found in the North American species Photinus pyralis, catalyzes the production of yellow-green light via oxidation of firefly luciferin in the presence of MgATP and molecular oxygen 1. Indeed, the light emitting property of luciferase-catalyzed reaction as well as its dependence on ATP makes this reaction a valuable tool in biological assays. The luciferase gene is commonly used as a reporter gene in order to visualize expression of various genes in a wide variety of organisms.
Through evolutionary history, bioluminenscence evolved via a number of different pathways in different organisms. Phylogenetically related species tend to share similar pathways of bioluminescence. For example, the luciferase of the Japanese firefly Luciola cruciata (PDB ID: 2D1S), shares about 98% sequence homology with that of the North American firefly (shown here)2. Indeed, both species emit yellow-green light, which is evidence that the functional domains of their respective luciferases are conserved.
Firefly luciferase is a 60,745.17 D, 550 residue, single-polymer protein, with a pI of 6.42, and is classified as a mono-oxygenase 3. The overall wild-type structure of the molecule is folded into two distinct domains. Residues 4-436 comprise the compact domain containing two β-sheets flanked on either side by α-helices and a distorted antiparallel β-barrel (divided into β-sheets A, B, and C). The C-terminus of the protein, consisting of residues 440-550, forms a small separate α+β domain. These secondary structures are assigned based on main-chain hydrogen bonding typical of folded proteins 1 .
The two Beta sheet subdomains in the N-terminal domain form a five-layered αβαβα tertiary arrangement, with two α helices sandwiched between the sheets and the other helices packed against the outer faces. These β-sheet subdomains have a similar topology. Each sheet consists of eight strands and displays the characteristic right-handed twist of parallel β-sheets as viewed along the strands. β-sheet A includes 3 antiparallel and 5 parallel β-strands along with six associated helices, and is constructed from a single section of the polypeptide comprised of residues 77-222, except for one beta strand and one alpha helix, which is positioned near residue 399 further down the polypeptide chain. β-sheet B is composed of two non-continuous segments of the chain at residues 22-70 and 236-351, and consists of two antiparallel and six parallel β-strands together with six α-helices. Each of the β-sheet cores consists of parallel strands joined to α-helices with regular right-handed crossover junctions. This results in the arrangement of helices on either side of the sheet antiparallel to the strands. These two β-sheets pack together, creating a long surface groove that is shaped by the C-termini of the strands and the N-termini of the helices lying between the sheets. The β-barrel subdomain, formed by three distinct faces, is present at one end of the groove, and serves to close it off. Two faces each consist of three-stranded antiparallel β-sheets, with one of the β-strands (2nd strand on β-sheet C) hydrogen bonding to the last strand of β-sheet B. The third side of the β-barrel is formed by the strands 7 and 8 of β-sheet B (numbered from N to C-terminal) and by the loop connecting them. Two shallow depressions on the concave surface of the molecule are formed by the packing of the β-barrel against the other two sheets. The groove consisting of the two β-sheets along with the two depressions on the concave surface form a system of valleys on the surface of the large domain opposite to the C-terminal domain 1.
The C-terminal domain is separated from the large N-terminal domain by a wide cleft, and is classified as an α+β structure. It contains two short β-strands in antiparallel fashion and a three-stranded mixed β-sheet with three helices packed at the sides. A lid is formed by the C-terminal domain over the β-barrel and the last two strands of the β-sheets on the N-terminal domain. The crystal structure of the hinge region connecting the two domains appears to be disordered, which means it is flexible 1.
Though much work still needs to be done to determine the exact location of the luciferase active site, putative locations of the active site have been proposed. The active site in firefly luciferase can be estimated from the position of residues that are highly conserved among families of related enzymes. Initial studies show that firefly luciferase is closely related to adenylation enzymes such as acyl-CoA ligases and peptide synthetases 1 . Several residues of firefly luciferase are absolutely conserved (Lys206, Glu 344, Asp422, Arg437, Gly446 and Glu455) in the enzyme superfamily, and are therefore likely to act on the binding of ATP and adenylate formation. The surface of the luciferase molecule contains distinct patches of conserved residues. These appear at the N-terminal and C-terminal domains facing each other across the cleft, and occur in short, highly conserved sequence motifs. For example, the signature motif for this enzyme superfamily, 198-[STG]-[STG]-G-[ST]-[TSE]-[GS]-x-[PALIVM]-K-206, is found in the loop connecting the antiparallel strands 6 and 7 of the β-sheet. This peptide contains the conserved Lys206 residues and is implicated in ATP binding. These conserved residues are located on opposite surfaces of the two domains joined by the cleft, or are present in the hinge. The cleft is too large to allow interaction of conserved surfaces and the substrate at the same time. Thus, it is likely that these two domains will come together and sandwich the substrate as the reaction proceeds, forming the active site 1. A later study illustrates that conformational changes during substrate binding in the S314-L319 loop conserved amongst luciferases may promote access of luciferin and the adenine ring of ATP in their binding pockets. In addition, various electrostatic and H-bond interactions with Mg2+, R218,F247, A348, H245, and K529 fix the position of luciferin in the binding pocket. As a result, one of the luciferin carboxylate oxygen atoms points toward the α-P of ATP, possibly promoting adenylate formation. The invariant R218 appears to form a favorable electrostatic interaction with the phenoxide ion of luciferin, and H245 possibly assists in positioning luciferyl-AMP primed to react with molecular oxygen 4. Some residues may be important in determining bioluminescence color. Studies show that specific mutations in firefly luciferase including H245N and S284T, can cause emission of red light and increased time of light emission 5,6. The change in color and enzyme kinetics indicates the vital role of these conserved residues in color determination. However, the exact nature of these color changes is still being elucidated.
In addition to the normal substrates, anesthetics such as bromoform can bind to the luciferin-binding pocket and can act as an inhibitor of luciferase activity. Two bromoform molecules appear close to one another and can bind to pockets in the large N-terminal domain. One is bound to the luciferin-binding site and interacts with the aliphatic chain of Arg337(shown here). The other is bound to a polar pocket next to the luciferin pocket that is accessible from the external surface. It interacts with Glu311, Glu354, Thr352, His310, and the guanidinum group of Arg337, causing minimal perturbations in order to coordinate itself to them (shown here). High concentrations of ATP appear to increase anesthetic binding, which is likely due to ATP binding to the enzyme that causes the two domains to come together. This may trap the bromoform molecules binding within the luciferin pocket, enhancing their binding affinities 7.
Though the structure of firefly luciferase has been well characterized, components of the luciferase active site as well as its interactions with various ligands are still being elucidated. Putative locations of the active site have been described, and many amino acid residues critical to luciferase function have been found. However, there is still much work to be done. As we move further into the century, these structural features and interactions will likely be clarified, expanding our knowledge of this useful enzyme.