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

Abstract

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the Cterminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the Cterminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the Nterminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.Key words: bioluminescence; chimeric protein; fatty acyl-CoA synthetase; firefly luciferaseFirefly luciferase catalyses the oxidation of firefly luciferin (D-luciferin) via an intermediate of luciferyl-AMP in the presence of O 2 , ATP, and Mg 2þ to emit light (Scheme 1). 1,2) When excess amounts of D-luciferin and ATP are present in the reaction mixture of firefly luciferase, a rapid inhibition of luminescence occurs.However, it is known that the addition of CoA to the reaction mixture prevents luminescence inhibition and enhances luminescence activity. 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. 13) Truncation analyses of the firefly luciferase gene indicated that removal of the N-terminal or the C-terminal amino acid residues results in significant loss of luminescence activity. For example, deletion of 12 amino acids at the C-terminus resulted in a 99.7% loss of activity by in vitro translation analysis. 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. NM 142964) encodes a fatty acyl-CoA synthetase, but not...

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

“…Thus, CG6178 is the first protein that was identified as a long-chain fatty acyl-CoA synthetase in Drosophila. To understand the catalytic properties, the chimeric proteins of P. pyralis luciferase and CG6178 were prepared, and only the chimeric protein of the N-terminal domain of P. pyralis luciferase and C-terminal domain of CG6178 showed the luminescence activity with *4% activity of P. pyralis luciferase [133].…”

“…The gene product of AbLL (AbLL) had medium-and long-chain fatty acylCoA synthetase activity, but not luciferase activity [136]. Thus, all homologous genes of firefly luciferase from luminous and non-luminous insects did not show the significant activity of luminescence [131][132][133][134][135][136]. As shown in Fig. 14, phylogenic analysis was performed using homologous genes from luminous and non-luminous insects.…”

“…The gene duplications might occur before luminous insects within Lampyridae, Phengodidae and Elateridae diverged [132][133][134][135][136][137].…”

Enzymatic promiscuity and the evolution of bioluminescence

“…Since Fluc shares sequence identity and enzymatic activity with insect ACSLs, Oba et al . tested whether chimeras between Fluc and CG6178 (a homologous ACSL from the fruit fly Drosophila melanogaster ) are capable of luciferase activity. The C‐terminal domain of CG6178 (residues 436–544) was connected to the N‐terminal domain of Fluc (residues 1–437) to make one of two chimeras.…”

Lessons Learned from Luminous Luciferins and Latent Luciferases

“…In addition to the latent luciferase activity of CG6178 and AbLL, weak activity of an ACSL with d -luciferin has been reported, and mutation of ACSLs can reveal nascent luciferase activity with d -luciferin . Furthermore, a chimera between firefly luciferase and CG6178 can be a functional luciferase, and chimeras between beetle luciferases can have high activity. , On the other hand, an ACSL from the beetle Pyrophorus angustus failed to demonstrate any luciferase activity despite its higher homology to beetle luciferases than CG6178 or AbLL . Potentially, chimeras between luciferases and ACSLs could also be used to modulate activity and substrate preference and to better understand the molecular basis for these effects.…”