Identification of paralogous genes of firefly luciferase in the Japanese firefly, Luciola cruciata

Oba, Yuichi; Sato, Mitsunori; Ohta, Yuichiro; Inouye, Satoshi

doi:10.1016/j.gene.2005.10.023

Cited by 26 publications

(57 citation statements)

References 17 publications

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“…All chemicals were obtained from commercial sources: [1][2][3][4][5][6][7][8][9][10][11][12][13][14] …”

Section: Methodsmentioning

confidence: 99%

“…We also identified that luciferase homologues isolated from the fruit fly Drosophila melanogaster (CG6178), the Japanese firefly L. cruciata (LcLL1), and the non-luminous beetle Tenebrio molitor (TmLL-1, TmLL-2, and TmLL-3) are a FACS [5][6][7][8]. Based on these results, we have been proposing that a luciferase (monooxygenase) arose from a FACS in insects [7].…”

Section: Introductionmentioning

confidence: 93%

“…Based on these results, we have been proposing that a luciferase (monooxygenase) arose from a FACS in insects [7]. Recently, we isolated a homologous gene of luciferase from non-luminous click beetle, Agrypnus binodulus, and the gene product was named AbLL [9].…”

Section: Introductionmentioning

confidence: 99%

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Functional conversion of fatty acyl‐CoA synthetase to firefly luciferase by site‐directed mutagenesis: A key substitution responsible for luminescence activity

2009

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a b s t r a c tWe demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis [Oba, Y., Ojika, M. and Inouye, S. (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett. 540,[251][252][253][254] and proposed that the evolutionary origin of beetle luciferase is a fatty acyl-CoA synthetase (FACS) in insect. In this study, we performed the functional conversion of FACS to luciferase by replacing a single amino acid to serine. This serine residue is conserved in luciferases and possibly interacts with luciferin. The mutants of FACSs in non-luminous click beetle Agrypnus binodulus (AbLL) and Drosophila melanogaster (CG6178) gave luminescence enhancement, suggesting that the serine residue is a key substitution responsible for luminescence activity.

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“…All chemicals were obtained from commercial sources: [1][2][3][4][5][6][7][8][9][10][11][12][13][14] …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

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Functional conversion of fatty acyl‐CoA synthetase to firefly luciferase by site‐directed mutagenesis: A key substitution responsible for luminescence activity

2009

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“…Luminescence assay was performed as described previously. 15,16,18) The reaction mixture (100 ml) contained D-luciferin (10 mM), ATP (250 mM), CoA (250 mM) and MgCl 2 (5 mM) in 100 mM Tris-HCl (pH 7.8). The reaction was started by the addition of the recombinant protein (0.1 mg) at 22-23 C. The light intensity was detected with an ATTO model AB-2200 luminometer (Tokyo) for 60 s. The luminescence spectrum was measured on a Jasco FP-777W fluorescence spectrophotometer (Jasco, Tokyo) at 25 C with the excitation light source turned off, and scan speed was 1,000 nm/min.…”

Section: Methodsmentioning

confidence: 99%

“…Adenylation activity for various fatty acids was determined with the formation of 32 P-AMP from [ -32 P]ATP by TLC analysis. 15,16,18) In brief, the reaction mixture (20 ml) containing fatty acid (10 mM), [ -32 P]ATP (0.33 mCi), ATP (250 mM), CoA (50 mM), MgCl 2 (5 mM), and purified Pp/Dm or Dm/Pp (50 nM) in 100 mM Tris-HCl (pH 7.8) was incubated at 25 C. After 30 min, the reaction was terminated by the addition of 20 mM of ethanol, of which 3 ml was spotted on a TLC plate. After development, the radioactivity of the produced 32 P-AMP was measured with an imaging analyzer model BAS 2500 (Fuji film, Tokyo).…”

Section: Methodsmentioning

confidence: 99%

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

Oba¹,

Tanaka²,

Inouye³

2006

Bioscience, Biotechnology, and Biochemistry

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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...

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Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

Inouye

2009

Cell. Mol. Life Sci.

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Firefly luciferase is a member of the acyl-adenylate/thioester-forming superfamily of enzymes and catalyzes the oxidation of firefly luciferin with molecular oxygen to emit light. Knowledge of the luminescence mechanism catalyzed by firefly luciferase has been gathered, leading to the discovery of a novel catalytic function of luciferase. Recently, we demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis from fatty acids in the presence of ATP, Mg(2+) and coenzyme A. Based on identification of fatty acyl-CoA genes in firefly, Drosophila, and non-luminous click beetles, we then proposed that the evolutionary origin of firefly luciferase is a fatty acyl-CoA synthetase in insects. Further, we succeeded in converting the fatty acyl-CoA synthetase of non-luminous insects into functional luciferase showing luminescence activity by site-directed mutagenesis.

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Identification of paralogous genes of firefly luciferase in the Japanese firefly, Luciola cruciata

Cited by 26 publications

References 17 publications

Functional conversion of fatty acyl‐CoA synthetase to firefly luciferase by site‐directed mutagenesis: A key substitution responsible for luminescence activity

Functional conversion of fatty acyl‐CoA synthetase to firefly luciferase by site‐directed mutagenesis: A key substitution responsible for luminescence activity

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

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

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