Mutagenesis Evidence that the Partial Reactions of Firefly Bioluminescence Are Catalyzed by Different Conformations of the Luciferase C-Terminal Domain

Abstract

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

“…Although firefly luciferase can adenylate both D-and L-luciferin (10), Nakamura et al recently showed that the L-luciferin adenylate inhibitor can be removed from the active site by thioesterification with CoA, whereas D-luciferin adenylate preferentially undergoes bioluminescent oxidation (39). Studies of the threedimensional structures of homologous AMP/CoA ligases also show that CoA is involved in important conformational changes during the second reaction in these enzymes, i.e., thioesterification in most ligases and oxygenation in beetle luciferases (40,41). It is remarkable that the removal of the C-terminal domain of firefly luciferase results in an impaired enzyme that produces weak red bioluminescence, indicating that the C-terminal is important for efficient yellow-green bioluminescence (42).…”

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

“…S7). The formation of a carbanion intermediate may be further facilitated in the enzyme by coordination of the substrate carbonyl oxygen to a conserved lysine residue (K443 in FLuc), which has been shown to be important for the oxidative reaction of LH 2 -AMP with firefly luciferase (18,31) and is found in all beetle luciferases and many ACSLs, including CG6178 (Fig. S1).…”

Catalytic Properties of Domain-Exchanged Chimeric Proteins between Firefly Luciferase andDrosophilaFatty Acyl-CoA Synthetase CG6178

“…Ayabe et al 22) suggested that the C-terminal domain of firefly luciferase plays an important role in stabilizing the complex of enzyme and luciferyl-AMP by analyses using the N-terminal domain of PpLase. By site-specific mutagenesis analyses, Branchini et al 7) proposed that Lys443 and Lys529 in the C-terminal domain of PpLase are essential in the catalytic steps of firefly luciferase as well as the other adenylate-forming enzymes. These two lysine residues are also conserved in CG6178.…”

“…3) To understand the catalytic properties, various analyses such as X-ray crystallography, 4,5) NMR, 6) site-directed mutagenesis, 7, and references therein) and gene truncation [8][9][10][11] were performed. Crystal structural analyses showed that firefly luciferase consists of a large N-terminal domain and a small C-terminal domain, 4,5) similar to acetyl-CoA synthetase 12) and fatty acyl-CoA synthetase.…”

“…8) Mutagenesis analyses have suggested the amino acid residues that participate in luminescence reaction. 7) Recently, we found that firefly luciferase is a bifunctional enzyme possessing luminescence and fatty acylCoA synthetic activities (Scheme 1). 14,15) Further, we determined that the homologous gene of firefly luciferase in Drosophila melanogaster (CG6178, GenBank accession no.…”