Research Article 287 Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes Elena Conti, Nick P Franks and Peter Brick* Background: Firefly luciferase is a 62 kDa protein that catalyzes the production Address: Biophysics Section, Blackett Laboratory, of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its Imperial College, London SW7 2BZ, UK. substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds *Corresponding author. through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that Key words: acyl-coenzyme A ligase, adenylate, utilize ATP in adenylation reactions. firefly luciferase, peptide synthetase, X-ray crystallography Results: We have determined the crystal structure of firefly luciferase at 2.0 Å Received: 30 Nov 1995 resolution. The protein is folded into two compact domains. The large N-terminal Revisions requested: 21 Dec 1995 domain consists of a b-barrel and two b-sheets. The sheets are flanked by Revisions received: 15 Jan 1996 a-helices to form an ababa five-layered structure. The C-terminal portion of the Accepted: 31 Jan 1996 molecule forms a distinct domain, which is separated from the N-terminal domain Structure 15 March 1996, 4:287–298 by a wide cleft. © Current Biology Ltd ISSN 0969-2126 Conclusions: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions. Introduction The firefly uses this enzyme to emit flashes of light to Bioluminescent organisms are widely distributed through- attract its mate. The enzyme is a 62 kDa molecular weight out terrestrial and aquatic environments and include lumi- oxygenase [2] but, unlike most other oxygenases, no redox nous bacteria, insects, marine coelenterates and crustacea prosthetic group is involved in the reaction. The enzyme [1]. The biological function of luminescence varies from requires ATP, molecular oxygen and the heterocyclic com- species to species, and ranges from distracting predators to pound firefly luciferin to generate light in a two-step attracting prey or mating partners. The production of light process [3] (Fig. 1). The reaction has unusual kinetics in arises from the conversion of chemical energy into an that firefly luciferase turns over very slowly [4]. After an excited electronic state, energetic enough to result in the initial flash of light, the luminescence rapidly decreases to emission of a photon of visible light. This is achieved by a low level of emission, probably due to product inhibition oxidation of a substrate (S) to an excited-state product of the enzyme. The quantum yield is the highest known (P*), which then decays to the ground state (P) emitting for any bioluminescent reaction [5], with nearly one photon light (hn). of light emitted for every luciferin molecule oxidized. S + O → P* → P + hn 2 Firefly luciferase has been extensively used in molecular The reaction is catalyzed by enzymes called luciferases. and cell biology, in particular for the efficient detection While luciferase enzymes from different species all utilize and quantification of ATP and as a reporter of genetic an oxidation reaction, these reactions involve a variety of function [6]. Luciferase has also been studied as a model different cofactors and unrelated substrates and proceed for possible protein–anaesthetic interactions, being one of through distinct reaction pathways. Indeed, the biological the few soluble proteins sensitive to a wide range of and biochemical diversity of the bioluminescent systems general anaesthetics [7]. suggests that the ability to make light arose from many separate origins during evolution [1]. The catalytic formation of an enzyme-bound adenylate intermediate is a common mechanism used by both Luciferase from the firefly Photinus pyralis (EC 1.13.12.7) is prokaryotic and eukaryotic organisms to activate sub- a peroxisomal protein found in the light-emitting organ strates. Enzymes as diverse as aminoacyl-tRNA syn- known as the lantern within the abdomen of the insect. thetases (aaRSs) and fatty-acyl coenzyme A (CoA) ligases 288 Structure 1996, Vol 4 No 3 Figure 1 Figure 2 The reaction catalyzed by firefly luciferase. In the first (activation) step, the substrate D-luciferin (Luc-COOH; 4,5-dihydro-2-[6-hydroxy-2-benzo- thiazolyl]-4-thiazolecarboxylic acid) is activated by acylation of its carboxylate group with the a-phosphate of ATP, resulting in the formation of an enzyme-bound luciferyl adenylate (Enzyme:Luc-CO-AMP) and release of pyrophosphate (PPi). In the second (oxidation) step, luciferyl adenylate reacts with stoichiometric amounts of molecular oxygen, via an a-peroxylactone intermediate [61], producing an enzyme-bound excited- state product (Enzyme:Luc=O*). This excited-stated product then decays to the ground state (oxyluciferin; Luc=O) emitting yellow-green light. link the carboxyl group of their substrates to the phospho- ryl moiety of AMP, and subsequently transfer the acti- vated substrate to an acceptor such as tRNA or CoA. The primary sequence of firefly luciferase is unrelated to that of the aaRSs, but shares extensive sequence similarity with acyl-CoA ligases [8] and with enzymes involved in the synthesis of linear and cyclic polypeptides in fungi and bacteria [9]. On the other hand, firefly luciferase shares no sequence homology with the light-emitting enzymes from bacteria, coelenterates and crustacea. Interest in luciferases was stimulated by Harvey’s discov- ery, 70 years ago [10], that virtually all bioluminescent reactions require oxygen. However, until quite recently, Ribbon representations of the firefly luciferase molecule shown in two little structural information was available on light-emitting orthogonal views. The three subdomains of the large N-terminal domain enzymes. Last year, Fisher and co-workers [11] deter- are shown in blue (b-sheet A), purple (b-sheet B) and green (b-barrel) and the small C-terminal domain is shown in yellow. Disordered loops mined the crystal structure of luciferase from the bac- are drawn in violet. (Generated using the program MOLSCRIPT [62].) terium Vibrio harveyi. This bacterial luciferase is a flavin monooxygenase that catalyzes the oxidation of a long- chain aldehyde and reduced flavin mononucleotide. We Results and discussion present the three-dimensional crystal structure of the The three-dimensional crystal structure was determined firefly luciferase from Photinus pyralis, which has been by the multiple isomorphous replacement (MIR) method, determined at 2 Å resolution. despite significant non-isomorphism upon binding of Research Article Crystal structure of firefly luciferase Conti, Franks and Brick 289 Figure 3 Stereo a-carbon trace viewed in a similar orientation to Figure 2a, with some sequence numbering for reference. heavy atoms. The structure was refined to 2 Å resolution consists of six parallel and two antiparallel b-strands and, with a crystallographic R-factor of 22.4% and a free together with six helices, is built up from two non-con- R-factor of 26.5%. The molecular model has good stereo- tiguous portions of the polypeptide chain: the polypeptide chemistry and includes 523 out of 550 residues. segments 22–70 and 236–351. Overall structure The core of the each b-sheet consists of parallel strands The firefly luciferase molecule folds into two distinct joined to a-helices with standard right-handed cross-over domains (Figs 2,3). The major portion of the structure, com- connections, resulting in the arrangement of helices on prising residues 4–436, consists of a compact domain con- either side of the sheet, antiparallel to the strands. A taining a distorted antiparallel b-barrel and two b-sheets, similar topology to that of the b-sheet core is present in which are flanked on either side by a-helices. The C termi- other proteins, as detected by comparison of the firefly nus of the protein (440–544) forms a small separate a+b luciferase molecule with known protein structures using domain. The secondary structure has been assigned on the the program DALI [15]. In particular, the superposition of basis of main-chain hydrogen bonding using the algorithm the chemotactic protein CheY [16] on b-sheet A results in of Kabsch and Sander [12]. Figure 4 shows a schematic 56 topologically equivalent a-carbons with an overall root representation of the molecular topology and the location of mean square (rms) separation of 1.8 Å. the subdomains in the primary sequence. N-terminal b-barrel subdomain N-terminal b-sheet subdomains The two b-sheets pack together and create a long surface The two b-sheet subdomains in the large N-terminal groove, shaped by the C termini of the strands and by the domain are assembled to form a five-layered ababa tertiary N termini of the helices between the sheets. The groove structure, so that two a-helices are sandwiched between the is closed at one end by the presence of the antiparallel sheets and the other helices are packed against the outer b-barrel. The barrel is distorted in that it is formed by faces. A similar arrangement has been reported for the three distinct faces. Two sides each consist of three- structures of 3-ketoacyl-CoA thiolase [13] and inositol stranded antiparallel b-sheets with one of the b-strands monophosphatase [14], but the topology and the relative (C2) also hydrogen bonding to the last strand of b-sheet B. orientation of the sheets is different in each case.